Highly purified and/or modified fucan compositions for the treatment of fibrous adhesions

ABSTRACT

Compositions, methods, systems, etc., are provided for modified and/or purified fucans and corresponding fucan-containing compositions that inhibit fibrous adhesions among other advantages. The purified/modified fucans and fucan compositions have a reduced level of non-fucan components or impurities such as those found in a starting fucan composition. Such reduced undesirable components or impurities include, for example, undesired components bound to the fucan and compounds in the composition that are not a part of or bound to the fucan.

CLAIM FOR PRIORITY

The present application claims the benefit of U.S. provisional patentapplication No. 62/711,364, filed Jul. 27, 2018; U.S. provisional patentapplication No. 62/711,372, filed Jul. 27, 2018; U.S. provisional patentapplication No. 62/711,335, filed Jul. 27, 2018; U.S. Provisional PatentApplication Ser. No. 62/713,399, filed Aug. 1, 2018; U.S. provisionalpatent application No. 62/722,135, filed Aug. 23, 2018; U.S. provisionalpatent application No. 62/755,311, filed Nov. 2, 2018; U.S. provisionalpatent application No. 62/793,514, filed on Jan. 17, 2019; U.S.provisional patent application No. 62/861,223, filed Jun. 13, 2019; U.S.Provisional Patent Application Ser. No. 62/713,392, filed Aug. 1, 2018;U.S. provisional patent application No. 62/713,413, filed Aug. 1, 2018;U.S. provisional patent application No. 62/722,137, filed Aug. 23, 2018;U.S. provisional patent application No. 62/755,318, filed on Nov. 2,2018; U.S. provisional patent application No. 62/861,228, filed Jun. 13,2019; U.S. Provisional Patent Application Ser. No. 62/755,328, filedNov. 2, 2018; U.S. provisional patent application No. 62/793,654, filedJan. 17, 2019; and, U.S. provisional patent application No. 62/861,235,filed Jun. 13, 2019, all of which applications are incorporated hereinby reference in their entirety.

BACKGROUND

Fucans (including fucoidan) are sulfated polysaccharides. In generalterms, this means that they are molecules made up of a number of sugargroups, and also have sulfur atoms attached to the sugar groups. Themain sugar group is called “fucose”, which is sugar that has 6 carbonatoms and has the chemical formula C₆H₁₂O₅. “Fucoidan” (or fucoidin)indicates fucans derived from brown algae (seaweed). Fucans can existalone, or in a mixture of other sugars, for example in a mixture ofsugars such as xylose, galactose, glucose, glucuronic acid and/ormannose. These other sugars can be extracted from the seaweed or othersource with the fucan. Although fucans are currently derived fromnatural sources such as the brown algae (seaweeds), sea cucumbers, etc.,mentioned herein, “fucan” includes polymer molecules having the chemicaland structural motifs of the fucans as discussed herein regardless ofthe ultimate source(s) of the fucans.

Fucoidan can be obtained from a variety of species of brown algaeincluding but not limited to: Adenocystis utricularis, Ascophyllumnodosum, Chorda filum, Cystoseirabies marina, Durvillaea antarctica,Ecklonia kurome, Ecklonia maxima, Eisenia bicyclis, Fucus evanescens,Fucus vesiculosis, Hizikia fusiforme, Himanthalia Elongata,Kjellmaniella crassifolia, Laminaria brasiliensis, Laminariacichorioides, Laminaria hyperborea, Laminaria japonica, Laminariasaccharina, Lessonia trabeculata, Macrocystis pyrifera, Pelvetiafastigiata, Pelvetia Canaliculata, Saccharina japonica, Saccharinalatissima, Sargassum stenophylum, Sargassum thunbergii, Sargassumconfusum, Sargassum fusiforme and Undaria pinnatifida. These exemplaryspecies are all from the taxonomic class Phaeophyceae and the majorityof these species fall into the families of Fucales and Laminariaceae.

Fucans including fucoidan have been shown to be efficacious in servingas a barrier device to prevent, inhibit, and treat the formation offibrous adhesions. They have also found use in the treatment of otherrelated diseases and conditions.

Thus, there has gone unmet a need for the preparation of purified fucansincluding such fucans being modified to have desired molecular weightdistributions and/or sulfate levels. The present compositions, systemsand methods, etc., provide these and/or other advantages.

SUMMARY

Compositions, methods, systems, etc., are provided for fucans andfucan-containing compositions that inhibit fibrous adhesions among otheradvantages. The fucans and fucan compositions herein comprise fucans,including purified/modified fucans, having particular levels of desired,specified fucan components. The body of this application often refers topurified/modified fucans and purified/modified fucan compositions; suchreferences include all fucans/fucan compositions herein except in theclaims or unless clearly limited to purified/modifiedfucans/purified/modified fucan compositions from the context. In someembodiments, the fucans and compositions containing them are suitablefor medical and surgical applications. The fucans and fucan compositionshave a reduced level of non-fucan components or impurities such as thosefound in a feedstock fucan composition. Such undesirable components orimpurities include, for example, undesired components that are bound tothe fucan (e.g., ionic, covalent, hydrogen bonding, etc.) and compoundsin the composition that are not a part of or chemically and/or ionicallybound to the fucan. The undesired fucan components can be quantified incomparison (e.g., w/w) to the fucan. The non-fucan compounds andundesired fucan components may hereafter collectively be referred to asimpurities of the fucan and/or of the compositions comprising the fucanherein. These purified/modified fucans can, for example, reducedangerous complications during the medical and surgical use of fucansdue to impurities.

The present compositions, methods, systems, etc., provide compositionscomprising desired fucans, including purified/modified fucans obtainedfrom starting or initial fucan compositions (i.e., fucan compositionsfrom which the purified/modified fucans can be derived; such startingfucan compositions may or may not be crude or have been previouslyprocessed, such as a feedstock fucan composition) as well as methods ofobtaining such desired purified/modified fucans and methods of use ofsuch purified/modified fucans.

In some aspects, the present systems, devices and methods, etc., providecompositions comprising fucans comprising a total content of fucose,galactose, sulfate and counterions of greater than 90% w/w, 92% w/w, 94%w/w, 95% w/w, 97% w/w, 97.5% w/w, 98%, 98.8%, 99%, 99.5%, or 99.9% w/w.In some embodiments, the fucose content can be greater than 25% w/w, 30%w/w, 35% w/w; the galactose content can be less than 10% w/w or 5% w/w.The total counterion content can be less than 17% w/w or 14% w/w. Thecounterion can be a pharmaceutically acceptable counterion, such asaluminum, arginine, benzathine, chloroprocaine, choline, sodium,potassium, lithium, ammonium, ethylene diamine, diethylamine,diethanolamine, ethanolamine, histidine, lysine, N-methyl glucamine,meglumine, procaine, triethylamine, zinc, calcium and magnesium. In someembodiments the counterion can comprise, consist of, or consistessentially of at least one of sodium and potassium.

In certain embodiments, the provide compositions comprising thepurified/modified fucans can comprise a total fucose, galactose andsulfate content of greater than 75% w/w, 80% w/w, 84% w/w. At least 60%w/w of the molecular weight distribution can be greater than 100 kDa, aweight average molecular weight greater than 100 kDa, and/or peakmolecular weight greater than 70 kDa. One exemplary approach fordetermining such distribution is to use an aqueous gel permeationchromatography set up comprising or consisting essentially of:

-   -   one 300 mm analytical gel permeation chromatography column with        a 7.8 mm inner diameter packed with hydroxylated        polymethacrylate-based gel, having an effective molecular weight        range of between about 50 kDa and about 5,000 kDa, one 300 mm        analytical gel permeation chromatography column with a 7.8 mm        inner diameter packed with hydroxylated polymethacrylate-based        gel, having an effective molecular weight range of between about        1 kDa and about 6,000 kDa and one 40 mm guard column with a 6 mm        inner diameter packed with hydroxylated polymethacrylate-based        gel, the two analytical gel permeation chromatography columns        and the one guard column contained in a column compartment at        about 30° C.;    -   a refractive index detector at about 30° C.;    -   0.1M sodium nitrate mobile phase run at 0.6 mL/min; and    -   quantification against a peak molecular weight standard curve        consisting essentially of a first dextran standard with a peak        molecular weight of about 2,200 kDa, a second dextran standard        with a peak molecular weight of between about 720 kDa and about        760 kDa, a third dextran standard with a peak molecular weight        between about 470 kDa and about 510 kDa, a fourth dextran        standard with a peak molecular weight between about 370 kDa and        about 410 kDa, a fifth dextran standard with a peak molecular        weight between about 180 kDa and about 220 kDa, and a sixth        dextran standard with a peak molecular weight between about 40        kDa and 55 kDa. The peak molecular weight standard curve further        can comprise a dextran standard with a peak molecular weight        between about 3 kDa and about 5 kDa.

The provide compositions comprising the fucans can have a number averagemolecular weight greater than 50 kDa, a sulfation level of between 20%w/w and 60% w/w or between 30% w/w and 55% w/w or 35% w/w and 52% w/w.

The total carbohydrate content can be between 27% w/w and 80% w/w. Thetotal fucose content as a percentage of the total carbohydrate contentcan be at least about 30% w/w, 50% w/w, 70% w/w, 90% w/w or 95% w/w. Thetotal galactose content as a percentage of the total carbohydratecontent can be below about 60% w/w or 20% w/w. The total of glucuronicacid, mannose, rhamnose, glucose and xylose content as a percentage ofthe total carbohydrate content can be below about 30% w/w.

Also provided herein are methods comprising making or using the providecompositions comprising the purified/modified fucans herein. The usingcan comprise treating fibrous adhesions.

In some embodiments, the fucan compositions herein include solid fucancompositions, and comprise at least 90% w/w fucans, such aspurified/modified fucans herein, the solid fucan composition furthercomprising a total water content of less than 7% w/w, 6% w/w, 5% w/w, 4%w/w, 3% w/w, 2% w/w, 1% w/w or 0.1% w/w.

In certain embodiments, the compositions herein are medically acceptablefucan compositions comprising a therapeutically effective amount of thepurified/modified fucans herein in a medically acceptable buffer ordiluent. The methods herein include treating a fibrous adhesion in ananimal such as a human comprising selecting purified/modified fucansherein to inhibit the fibrous adhesion and administering atherapeutically effective amount of the composition or the fucan withinthe composition at a dosage range between 0.5 mg/kg and 50 mg/kgcompositions herein to the site of a wound of the animal. The dosagerange can also be between 1 mg/kg and 25 mg/kg. Methods can alsocomprise use of a dosage range comprising between 0.5 mg/kg and 50mg/kg, or between 1 mg/kg and 25 mg/kg, to treat a targeted disease ordisorder, including for example fibrous adhesions.

Also provided herein are methods of making the compositions comprisingpurified/modified fucans herein including, for example, methods forremoving impurities from a starting fucan composition, such as afeedstock fucan composition, to obtain purified/modified fucans. Suchmethods can comprise, for example:

-   -   providing a starting fucan composition comprising impurities;    -   adding a flocculation aid to the starting fucan composition to        produce a reaction mixture;    -   flocculating the impurities by heating the reaction mixture to        produce flocculated impurities; and    -   removing the flocculated impurities.

Providing the starting fucan compositions can comprise providing thestarting fucan compositions as a solution, and the methods further cancomprise collecting the purified/modified fucans in a reduced-impuritiessolution. The flocculating the impurities can comprise heating thereaction mixture in excess of atmospheric pressure, and the flocculationaid can comprise a salt, such as chloride, bromide, iodide, fluoride,sulfate, sulfite, carbonate, bicarbonate, phosphate, nitrate, nitrite,acetate, citrate, silicate and/or cyanide of an alkali metal, alkalineearth metal, aluminum and/or ammonium. The flocculation aid can comprisea base such as hydroxide and/or oxide of an alkali metal, alkaline earthmetal, aluminum and/or ammonium.

The impurities removed comprise at least one of particulates, lipids,fatty acids, phlorotannins, laminarins, alginates, proteins, Maillardreaction products, fucoxanthin, chlorophyll, bacteria, cellularcomponents and DNA.

Additional methods for removing impurities from starting fucancompositions to obtain purified/modified fucans can comprise:

-   -   providing starting fucan compositions as a solid and an        extraction media incapable of dissolving fucans, configured for        dissolving impurities;    -   mixing the starting fucan compositions with the extraction media        to produce a mixture of the purified/modified fucans and the        extraction media; and    -   separating the purified/modified fucans from the extraction        media.

The methods can further comprise collecting the compositions comprisingthe purified/modified fucans as a solid. The extraction media cancomprise at least one organic solvent with a relative polarity less than0.765. The values for relative polarity can be normalized frommeasurements of solvent shifts of absorption spectra. See for exampleChristian Reichardt, Solvents and Solvent Effects in Organic Chemistry,Wiley-VCH Publishers, 3rd ed., 2003. The organic solvent can comprise atleast one of ethanol, isopropanol, methanol, benzene, diethyl ether,decamethylcyclo-pentasiloxane, ethyl acetate, butanol, hexane, heptane,heptanol, octanol and decanol. The extraction media further can compriseat least one of a base, a detergent and an oxidizing agent. Providingthe starting fucan compositions in a solid form can compriseprecipitating the starting fucan compositions from a solution. Theimpurities removed can comprise at least one of particulates, lipids,fatty acids, phlorotannins, laminarins, alginates, proteins, Maillardreaction products, fucoxanthin, chlorophyll, bacteria, cellularcomponents and DNA.

Further methods for removing impurities from starting fucan compositionsto obtain purified/modified fucans can comprise:

-   -   providing starting fucan compositions comprising impurities,        including suspended impurities in a solution;    -   precipitating the impurities from the solution using an        ionic-multivalent impurity precipitant, thereby producing a        mixture of suspended impurities, precipitated impurities and a        supernatant solution; and    -   separating the suspended impurities and precipitated impurities        from the supernatant solution.        The methods can also comprise collecting the supernatant        solution comprising the compositions comprising the        purified/modified fucans.

The ionic-multivalent impurity precipitant can comprise a salt of adivalent or trivalent cation; the salt can be a chloride, bromide,iodide, fluoride, sulfate, sulfite, carbonate, bicarbonate, phosphate,nitrate, nitrite, acetate, citrate, silicate and/or cyanide. The cationcan be an alkaline earth metal, zinc, aluminum, copper and/or iron. Theionic-multivalent impurity precipitant can comprise a base of a divalentor trivalent cation. The base can be a hydroxide and/or oxide of analkaline earth metal, zinc, aluminum, copper and/or iron.

The separating the suspended impurities and precipitated impurities fromthe supernatant solution can further comprise flocculating the suspendedimpurities and precipitated impurities by adding a flocculant to themixture of suspended impurities, precipitated impurities and supernatantsolution. The flocculant can comprise at least one of potassium aluminumsulfate; sodium aluminum sulfate; ammonium aluminum sulfate; calciumchloride; sodium phosphate; aluminum hydroxide; aluminum chloride;ferric chloride; ferric sulfate; ferrous sulfate; sodium silicate;calcium silicate; calcium phosphate; zinc chloride; calcium carbonate;calcium bicarbonate; potassium sulfate; magnesium phosphate;acrylamides; acrylic acid; aluminum chlorohydate; polyaluminiumchloride; tannins; formaldehyde; melamine; N,N-dimethylaminoethylacrylate methyl chloride; N,N-dimethylaminoethyl methacrylate methylchloride quaternary; and polydiallyldimethyl-ammonium chloride. Themethods further can comprise maintaining a pH of between about 7 and 14.Maintaining the pH can comprise the addition of base. The impuritiesremoved can comprise at least one of particulates, lipids, fatty acids,phlorotannins, laminarins, alginates, proteins, Maillard reactionproducts, fucoxanthin, chlorophyll, bacteria, cellular components andDNA.

Methods for removing impurities from starting fucan compositions toobtain purified/modified fucans can comprise:

-   -   providing starting fucan compositions comprising impurities;    -   adjusting the starting fucan compositions pH to between about 8        and 14;    -   adding to the starting fucan compositions a cellular disrupting        agent configured for lysing cellular components to produce a        reaction mixture comprising the cellular disrupting agent,        biomolecular lysates and the starting fucan compositions; and    -   removing the cellular disrupting agent and biomolecular lysates        from the reaction mixture.

Providing the starting fucan compositions can comprise providing thestarting fucan compositions as a solution, and the methods further cancomprise collecting the compositions comprising the purified/modifiedfucans as a solid or in a reduced-impurities solution. The cellulardisrupting agent can comprise a detergent, which can be an anionicdetergent, a cationic detergent, or a non-ionic detergent. The detergentcan comprise at least one of sodium dodecyl sulfate (SDS), benzalkoniumchloride, Triton X 100®, Triton X 114®, Brij® detergents, Tween®detergents, sodium deoxycholate, and alkylbenzenesulfonates. Removingthe cellular disrupting agent and biomolecular lysates can compriseadding to the reaction mixture a flocculant configured for flocculatingthe cellular disrupting agent and biomolecular lysates. Removing thecellular disrupting agent can comprise adding to the reaction mixture aprecipitant configured for rendering the cellular disrupting agentinsoluble in the reaction mixture, producing precipitates. Removing thebiomolecular lysates can comprise adding to the reaction mixture aprecipitant configured for rendering the biomolecular lysates insolublein the reaction mixture, producing precipitates. The methods further cancomprise adding to the reaction mixture a flocculant configured forflocculating the precipitates. The flocculant can comprise at least oneof potassium aluminum sulfate; sodium aluminum sulfate; ammoniumaluminum sulfate; calcium chloride; sodium phosphate; aluminumhydroxide; aluminum chloride; ferric chloride; ferric sulfate; ferroussulfate; sodium silicate; calcium silicate; calcium phosphate; zincchloride; calcium carbonate; calcium bicarbonate; potassium sulfate;magnesium phosphate; acrylamides; acrylic acid; aluminum chlorohydate;polyaluminium chloride; tannins; formaldehyde; melamine;N,N-dimethylaminoethyl acrylate methyl chloride; N,N-dimethylaminoethylmethacrylate methyl chloride quaternary; andpolydiallyldimethyl-ammonium chloride.

Removing the anionic detergent can comprise anionic adsorption; removingthe cationic detergent can comprise cationic adsorption; removing thenon-ionic detergent can comprise micellar phase separation; and,removing the detergent can comprise hydrophobic adsorption. Removing thedetergent can comprise:

-   -   diluting the reaction mixture until the concentration of the        detergent can be below a predetermined concentration; and    -   subjecting the reaction mixture comprising the detergent to        diafiltration over a tangential flow filtration filter with a        molecular weight cut-off above the largest molecular weight of        the detergent.

The methods can also comprise adding a chelating agent to the reactionmixture after providing the starting fucan compositions and beforeremoving the cellular disrupting agent. The chelating agent can compriseethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanol,ethylene diamine, porphine and/or citric acid, and the methods furthercan comprise adding an oxidant-quenching agent to the reaction mixturebefore removing the cellular disrupting agent to quench oxidants in thereaction mixture, or adding a bacteriostatic agent to the reactionmixture after providing the starting fucan compositions and beforeremoving the cellular disrupting agent. The bacteriostatic agent cancomprise sodium sulfite, ethylenediaminetetraacetic acid (EDTA),benzalkonium chloride, ethanol, and/or thiourea. The impurities removedcan comprise at least one of particulates, lipids, fatty acids,phlorotannins, laminarins, alginates, proteins, Maillard reactionproducts, fucoxanthin, chlorophyll, bacteria, cellular components andDNA.

Methods for removing impurities from starting fucan compositions toobtain the compositions comprising the purified/modified fucans can alsocomprise:

-   -   providing starting fucan compositions comprising impurities in        an aqueous starting solution;    -   mixing the aqueous starting solution with an organic solvent to        produce an aqueous-organic phase mixture; and    -   separating the aqueous-organic phase mixture to obtain an        aqueous portion and an organic portion.

The methods further can comprise collecting the aqueous portioncomprising the purified/modified fucans. The organic solvent cancomprise at least one organic solvent with a relative polarity less than0.765, which can be at least one of ethanol, isopropanol, methanol,benzene, decamethylcyclo-pentasiloxane, ethyl acetate, hexane, heptanol,octanol, decanol, heptane, isobutyl acetate, anisole, isopropyl acetate,1-butanol, butyl acetate, methylisobutylketone, pentane, 1-pentanol,ethyl ether, and propyl acetate. The impurities removed can comprise atleast one of particulates, lipids, fatty acids, phlorotannins,laminarins, alginates, proteins, Maillard reaction products,fucoxanthin, chlorophyll, bacteria, cellular components and DNA.

Methods for modifying the cationic content of starting fucancompositions can comprise:

-   -   providing starting fucan compositions in a starting solution;        and    -   diafiltering the starting solution across a tangential flow        filtration filter with a solution of a chelating agent across a        tangential flow filtration filter to produce a retentate fucan        compositions.

The chelating agent can comprise at least one ofethylenediamine-tetraacetic acid (EDTA), 2,3-dimercapto-1-propanol,ethylene diamine, porphine or citric acid. The retentate fucancompositions can comprise a cationic content consisting essentially ofsodium and/or potassium.

Methods for removing impurities from starting fucan compositions toobtain purified/modified fucans can further comprise:

-   -   providing starting fucan compositions comprising impurities;    -   subjecting the starting fucan compositions to a suitable        pressure above 70 bar and a suitable temperature above 30° C. in        a supercritical extractor; and    -   filling the supercritical extractor with a supercritical fluid        to remove impurities into the supercritical fluid; and    -   removing the supercritical fluid containing the extracted        impurities after a predetermined amount of time.

The methods further can comprise collecting the purified/modified fucansremaining in the supercritical extractor; the pressure can be betweenabout 70 bar and about 2000 bar, and the temperature can be betweenabout 30° C. and about 300° C. The starting fucan compositions can be aliquid or a solid. The supercritical fluid can comprise at least one ofcarbon dioxide, ethanol, ethane, hydrochloric acid, hydrofluoric acid,sulfuric acid and nitric acid. The predetermined amount of time can bebetween about 5 minutes and 50 hours. The impurities removed cancomprise at least one of particulates, lipids, fatty acids,phlorotannins, laminarins, alginates, proteins, Maillard reactionproducts, fucoxanthin, chlorophyll, bacteria, cellular components andDNA.

These and other aspects, features and embodiments are set forth withinthis application, including the following Detailed Description andattached drawings. Unless expressly stated otherwise, all embodiments,aspects, features, etc., can be mixed and matched, combined and permutedin any desired manner.

These and other aspects, features and embodiments are set forth withinthis application, including the following Detailed Description andattached drawings. Unless expressly stated otherwise, all embodiments,aspects, features, etc., can be mixed and matched, combined and permutedin any desired manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts an exemplary system for modifying thecationic content of a starting fucan composition and removing lowmolecular weight non-fucan components using a chelating agent intangential flow filtration.

FIG. 2A depicts NMR results demonstrating that certain fucans treatedaccording to methods herein undergo structural changes to the fucans.

FIG. 2B depicts 2-D NMR results demonstrating that certain fucanstreated according to methods herein undergo structural changes to thefucans.

The drawings present exemplary embodiments of some aspects of thepresent compositions, methods, etc. Embodiments of the systems, methods,etc., herein may include further features or steps not shown in thedrawings. Further, the exemplifications set out herein illustrateembodiments of the systems, methods, etc., in one or more forms, andsuch exemplifications are not to be construed as limiting the scope ofthe disclosure in any manner. The embodiments herein are not exhaustiveand do not limit the disclosure to the precise form disclosed, forexample in the following detailed description.

DETAILED DESCRIPTION

The current compositions, systems, methods, etc., presented hereincomprise purified/modified fucans. The present compositions can beeffective for medical treatments, post-surgical treatments, diseaseinhibition etc. In some embodiments, the fucan is fucoidan. The presentpurified/modified fucans can themselves be, or can be included on or in,medical devices, medical materials, combination products or inpharmaceutically acceptable, therapeutically and/or medically effectivecompositions.

The following paragraphs turn to a brief general discussion of some ofthe compositions herein comprising purified/modified fucans, includingthose that can be created using the methodologies discussed herein fromstarting fucan compositions via various methods that can be performedusing any suitable reaction mixture such as solutions, suspensions,solids, gels or other modalities depending on the chosen method(s).

Compositions

The current compositions, systems, etc., presented herein provide, incertain embodiments, medically acceptable purified/modified fucancompositions comprising therapeutically effective amounts ofpurified/modified fucan compositions for the treatment of fibrousadhesions, such as surgical adhesions, as well as arthritis, psoriasisor other diseases as desired. The purified/modified fucans in thecompositions can comprise more than about 75% w/w total fucose,galactose and sulfate, for example more than about 80% w/w and more than84% w/w total fucose, galactose and sulfate. In some embodiments, thepurified/modified fucans may further comprise up to at least about 5%,7%, 9%, 10% or 11% w/w of at least one counterion. In some embodiments,the counterion is a pharmaceutically acceptable counterion. In someembodiments, the counterion is ionically bound to the sulfate grouppresent on the fucan. Pharmaceutically acceptable counterions mayinclude at least one of aluminum, arginine, benzathine, chloroprocaine,choline, sodium, potassium, lithium, ammonium, ethylene diamine,diethylamine, diethanolamine, ethanolamine, histidine, lysine, N-methylglucamine, meglumine, procaine, triethylamine, zinc, calcium andmagnesium. The sulfur containing component of fucan is bound via a C—O—Slinkage. The oxygen in such linkage can be considered primarily bound toeither the carbon or the sulfur depending on various factors. The term“sulfate” as used herein refers to both embodiments.

In certain embodiments, the purified/modified fucans herein comprise atleast about 85% w/w, 90% w/w, 94% w/w, 97% w/w or 98% w/w fucose,galactose, sulfate, and counterions. Exemplary counterions include up toabout 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% w/w calcium,magnesium, potassium and/or sodium. In some embodiments, thepurified/modified fucan comprises at least about 25%, 30% or 35% w/wfucose. In some embodiments, the purified/modified fucan comprises lessthan about 10%, 5% or 4% w/w galactose. In some embodiments, thepurified/modified fucan consists essentially of, or consists of, suchtotal of fucose, galactose, sulfate and counterion components. In someembodiments, the fucans herein substantially or completely lack allsugar components other than the fucose and galactose. In someembodiments, the fucans herein substantially or completely lack one ormore of glucuronic acid, mannose, rhamnose, xylose, galactose, orglucose; as used in this sentence, “substantially lacks” indicates thatthe presence, if any, of such sugar components is low enough that thepresence is pharmaceutically and medically immaterial.

In some further embodiments, the purified/modified fucans herein can beused for a plurality of applications, including the inhibition,prevention, removal, reduction, or other treatment of fibrous adhesionsand other targets and other diseases and/or conditions. Treatmentincludes that the fucans reduce or prevent the development of a targetdisease or other condition, such as reducing or preventing the formationof fibrous adhesions at a target site, which is typically a selectedtarget site identified by a surgeon or other practitioner as comprisingor reasonably susceptible to having fibrous adhesions (or other diseasesor conditions), and also includes elimination of existing diseases orother conditions, including for example the elimination ofalready-existing fibrous adhesions. For such inhibition, prevention,removal, reduction, or other treatment, the fucan is typically providedin a medically acceptable medical device, combination product, orpharmaceutically effective composition that contains additionalcomponents such as binders, adjuvants, excipients, etc., as well as, ifdesired, additional medically active substances such as secondary drugsthat are contained within the composition but not attached to the fucan,and/or that can be attached to the fucan.

In still further embodiments, the compositions comprising thepurified/modified fucans herein can be solids, for example solidcompositions comprising a water content of less than about 7% w/w, forexample less than about 6%, 5% w/w, 4% w/w, 3% w/w or 2% w/w watercontent.

The molecular weight distribution of the purified/modified fucans may bemeasured using any desired, appropriate measurement system. Differentsystems can yield different readings or results from differentcompositions having essentially the same make-up, or even from the samebatch when measured differently. One suitable measurement system is anaqueous gel permeation chromatography set up consisting essentially ofone 300 mm analytical gel permeation chromatography column with a 7.8 mminner diameter packed with hydroxylated polymethacrylate-based gel,having an effective molecular weight range of between about 50 kDa andabout 5,000 kDa, one 300 mm analytical gel permeation chromatographycolumn with a 7.8 mm inner diameter packed with hydroxylatedpolymethacrylate-based gel, having an effective molecular weight rangeof between about 1 kDa and about 6,000 kDa and one 40 mm guard columnwith a 6 mm inner diameter packed with hydroxylatedpolymethacrylate-based gel, the two analytical gel permeationchromatography columns and the one guard column contained in a columncompartment at about 30° C., a refractive index detector at about 30°C., 0.1M sodium nitrate mobile phase run at 0.6 mL/min, andquantification against a peak molecular weight standard curve consistingessentially of a first dextran standard with a peak molecular weight ofabout 2,200 kDa, a second dextran standard with a peak molecular weightof between about 720 kDa and about 760 kDa, a third dextran standardwith a peak molecular weight between about 470 kDa and about 510 kDa, afourth dextran standard with a peak molecular weight between about 370kDa and about 410 kDa, a fifth dextran standard with a peak molecularweight between about 180 kDa and about 220 kDa, and a sixth dextranstandard with a peak molecular weight between about 40 kDa and 55 kDa.The peak molecular weight standard curve may further comprise a dextranstandard with a peak molecular weight between 3 kDa and 5 kDa.

The purified/modified fucans herein can have a molecular weightdistribution wherein at least about 25%, 30%, 40%, 50%, 60%, 70%, 75%,90%, 92%, 97% or 98% w/w of the distribution is greater than 100 kDa.The purified/modified fucans herein may comprise fucans with a molecularweight distribution wherein at least about 50%, 60%, 70%, 80%, or 90%w/w of the distribution is greater than 200 kDa. The purified/modifiedfucans herein can have a molecular weight distribution wherein at leastabout 25%, 30%, 40%, 50%, 60%, 70%, or 75% w/w of the distribution isgreater than 500 kDa. The purified/modified fucans herein can have amolecular weight distribution wherein at least about 5%, 10%, 20%, 30%,or 40% w/w of the distribution is greater than 1600 kDa.

The purified/modified fucans herein can have a weight average molecularweight greater than about 100 kDa, for example between about 100 kDa andabout 10,000 kDa, between about 200 kDa and about 8,000 kDa, betweenabout 350 kDa and about 8,000 kDa, between about 450 kDa and about 8,000kDa, between about 580 kDa and about 8,000 kDa, or between about 800 kDaand about 2,000 kDa. The purified/modified fucans herein can have a peakmolecular weight greater than about 70 kDa, for example between about 70kDa and about 1200 kDa, between about 100 kDa and about 1200 kDa,between about 200 kDa and about 1200 kDa, between about 400 kDa andabout 1200 kDa, or between about 400 kDa and about 900 kDa.

The purified/modified fucans herein can have a number average molecularweight greater than about 50 kDa, between about 50 kDa and about 1,000kDa, between about 70 kDa and about 1000 kDa, between about 150 kDa andabout 1000 kDa, between about 250 kDa and about 1000 kDa, or betweenabout 250 kDa and about 700 kDa.

The purified/modified fucans herein can have a sulfation level ofbetween about 10% w/w and 70% w/w, between about 20% w/w and 65% w/w,between about 30% w/w and 60% w/w, or between about 40% w/w and 60% w/w.

The purified/modified fucans herein can have a molar ratio of totalfucose:total sulfate of between about 1:0.5 and 1:4, between about 1:0.8and 1:3.5, between about 1:1 and 1:2.5, between about 1:1.2 and 1:2.0,or between about 1:1.5 and 1:3. Purified/modified fucans herein can havea molar ratio of total fucose plus galactose:total sulfate of betweenabout 1:0.5 and 1:4, between about 1:0.8 and 1:3.5, between about 1:1and 1:2.5, between about 1:1.2 and 1:2.0, or between about 1:1.5 and1:3.

The purified/modified fucans herein can have a total carbohydratecontent of between about 27% w/w and 70% w/w, between about 30% w/w and80% w/w, between about 40% w/w and 90% w/w, or between about 50% w/w and96% w/w. The purified/modified fucans herein can have a fucose contentas a percentage of total carbohydrate of about 30% w/w and 100% w/w,between about 40% w/w and 95% w/w, or between about 50% w/w and 90% w/w.The fucans herein may have a galactose content as a percentage of totalcarbohydrate of 0% w/w and 60% w/w, between about 5% w/w and 30% w/w, orbetween about 8% w/w and 10% w/w. The fucans herein may have aglucuronic acid content as a percentage of total carbohydrate contentbetween about 0% w/w and 10% w/w, a mannose content as a percentage oftotal carbohydrate content between about 0% w/w and 7% w/w, a rhamnosecontent as a percentage of total carbohydrate content between 0% w/w and4% w/w, and a xylose content as a percentage of total carbohydratecontent between 0% w/w and 20% w/w. The fucans herein may have a totalglucuronic acid, mannose, rhamnose, glucose and xylose content of lessthan about 30% w/w, or less than about 12% w/w.

In some embodiments, the purified/modified fucans herein, when dissolvedat a concentration of 50 mg/mL in water, have a viscosity of betweenabout 4 cP and about 50 cP, between about 5 cP and about 40 cP, betweenabout 10 cP and about 30 cP, about 15 cP, about 20 cP and about 25 cP.In certain embodiments, the purified/modified fucans herein, whendissolved in water at 1 mg/mL through 100 mg/mL form a solution that isone of clear and colorless, clear and light yellow or clear and lightbrown.

The purified/modified fucans herein can be provided in a paste, gel,patch, film, spray, liquid, lotion, cream, solution, suspension, solid,implant, microsphere or other desired form.

The compositions presented herein can be a solid consisting essentiallyof the purified/modified fucans. The purified/modified fucan may consistessentially of fucose, galactose, sulfate and counterions.

The purified/modified fucans herein can be in a solution comprisingbetween about 0.01 mg/mL and about 300 mg/mL of fucan, for examplebetween about 0.1 mg/mL and about 100 mg/mL, between about 1 mg/mL andabout 50 mg/mL and between about 20 mg/mL and about 80 mg/mL. The fucanmay consist essentially of fucose, galactose, sulfate and counterions.

The purified/modified fucans can be in a gel comprising between about100 mg/mL and about 1000 mg/mL of fucan, for example between about 100mg/mL and about 500 mg/mL and between about 300 mg/mL and about 800mg/mL. The fucan may consist essentially of fucose, galactose, sulfateand counterions.

The purified/modified fucans can be in a film comprising between about100 mg/mL and about 1000 mg/mL of fucan, for example between about 100mg/mL and about 500 mg/mL and between about 300 mg/mL and about 800mg/mL. The fucan may consist essentially of fucose, galactose, sulfateand counterions.

The purified/modified fucans herein can be administered as a componentof a medical device, combination product and/or pharmaceuticalcomposition comprising any number of pharmaceutically acceptableexcipients, for example, gelatin, hypromellose, lactose, water forinjection USP, sodium chloride, sodium phosphate, sodium citrate, sodiumascorbate, phosphate buffers, citrate buffers, phosphate-citratebuffers, pluronic, cellulose, alginate, acrylate, hyaluronic acid,polyethylene glycol, chitosan, injectable excipient and lactatedRinger's injection USP.

The purified/modified fucans herein can be administered as a paste, gel,patch, film, spray, liquid, lotion, cream, solution, suspension, solid,implant, microsphere or other desired form.

The purified/modified fucans can be administered via intravenous,intraarticular, intralesional, intravaginal, rectal, intramuscular,intraperitoneal, subcutaneous, topical, intranasal, intraocular or oraladministration routes. The purified/modified fucans can be directlydelivered to the disease site. The purified/modified fucans can becontinuously released to the disease site via controlled release from apolymeric dosage form.

The purified/modified fucans herein can be administered as a componentof a pharmaceutical composition comprising the purified/modified fucansand at least one other drug. The drug can be at least one of paclitaxel,doxorubicin, camptothecin, etoposide, mitoxantrone, methotrexate,menadione, plumbagin, juglone, beta-laperchone cyclosporin,sulfasalazine, steroid, rapamycin, retinoid, docetaxel, colchicine,antisense oligonucleotide and ribozyme.

In certain embodiments, the purified/modified fucans herein can have anacetyl content of less than about 5% w/w, less than about 2% w/w, andabout 0% w/w. In some embodiments, the purified/modified fucans hereincomprise substantially 0% w/w acetyl content when measured by 2D ¹H-¹³Cheteronuclear multiple quantum coherence at 70° C. with solvent signalsuppression on a 600 MHz spectrometer equipped with 5-mm cold probe, inthe range from 10-30 ppm in the carbon dimension, in 8 increments of256-512 scans each.

Methods

Methods, systems, etc., are provided for purifying and/or modifying afucan, for example from a starting fucan composition comprising fucans,for example a feedstock fucan composition, or other fucan-containingcompositions. “Impurities” as used herein refers to any component of thefucan that is not fucose, galactose, sulfate or a counterion, and to anynon-fucan component or compound or substance present in a compositioncomprising a fucan. Impurities can be bound to the fucan, for example,proteins ionically and/or chemically bound to the fucan, sugar residuesother than fucose and galactose that are part of the fucan polymericstructure, other saccharides chemically bound to the fucan, andnon-fucan impurities that are not bound to the fucan but are present ina starting fucan composition such as a feedstock fucan composition.Examples of such impurities include but are not limited to particulates,lipids, fatty acids, phlorotannins, laminarins, alginates, proteins,Maillard reaction products, fucoxanthin, chlorophyll, bacteria, cellularcomponents and DNA, several of which contain chromophores and result inthe presence of brown, yellow and green colors in starting fucancompositions and several of which can be ionically and/or chemicallybound to or part of the fucan in the starting fucan composition. Incertain embodiments, the methods, etc., herein can be used to preparepurified/modified fucans comprising at least about 88% w/w, 89% w/w, 90%w/w, 91% w/w, 92% w/w, 93% w/w, 94% w/w, 95% w/w, 96% w/w, 97% w/w,97.1% w/w, 98% w/w, 98.8% w/w, 99% w/w, 99.5% w/w, or 99.9% w/w offucose, galactose, sulfate and counterions. In some embodiments, thepurified/modified fucan comprises at least about 75%, 78%, 80%, 82% or84% w/w fucose, galactose and sulfate. In some embodiments, thepurified/modified fucan comprises less than about 0.1%, 0.5%, 1%, 2.9%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12% w/w impurities. Some ofthese impurities may cause dangerous complications upon the medicaland/or surgical use of fucans.

In some embodiments, the current disclosure presents purified/modifiedfucans with low levels of impurities that are suitable for medical andsurgical applications, for example, the prevention of fibrous adhesions.

The following paragraphs turn to a brief general discussion of some ofthe methodologies that can be used to create the purified/modifiedfucans herein.

Physically Induced Flocculation

A starting fucan composition, such as a feedstock fucan composition,comprising high levels of impurities undergoes a flocculation ofimpurities, which can be a physically-induced flocculation. The methodcan comprise: providing a starting fucan composition; adding aflocculation aid to the starting fucan composition to produce a reactionmixture; flocculating the impurities in the starting fucan compositionby heating the reaction mixture; separating the flocculated impuritiesfrom the reaction mixture; and collecting the desired purified/modifiedfucan after the separating.

Flocculating the impurities by heating the reaction mixture may compriseheating the reaction mixture while subjecting the reaction mixture to apressure in excess of atmospheric pressure. Suitable flocculation aidsinclude without limitation, salts and/or bases, for example chlorides,bromides, iodides, fluorides, sulfates, sulfites, carbonates,bicarbonates, phosphates, nitrates, nitrites, acetates, citrates,silicates, oxides, hydroxides and/or cyanides of an alkali metal,alkaline earth metal, aluminum and/or ammonium, for example, sodiumchloride, sodium sulfate, potassium chloride, calcium sulfate, sodiumphosphate, sodium nitrate, lithium chloride, lithium nitrate, ammoniumchloride, sodium carbonate, sodium hydroxide. Separating the flocculatedimpurities from the reaction mixture may comprise one or more ofcentrifuging, filtering, sedimentation or hydrodynamic flow separationof the reaction mixture.

The methods, etc., herein may further comprise desalting the startingfucan composition before adding a flocculation aid. The desalting maycomprise diafiltrating the starting fucan composition as a solution inwater across a molecular weight cutoff (MWCO) tangential flow filtration(TFF) filter. The diafiltrating may comprise diafiltrating the startingfucan composition with distilled water. The molecular weight cutoff TFFfilter can have a molecular weight cutoff smaller than a desiredmolecular weight separation point or target in or for thepurified/modified fucan, for example a 50 kDa, 70 kDa, 100 kDa, 200 kDa,300 kDa, 500 kDa or 1000 kDa molecular weight cut-off.

The method can be performed in basic and neutral environments. Theadding of a flocculation aid to the starting fucan composition maytherefore comprise rendering the starting fucan composition basic toprevent or inhibit the fucan in the starting fucan composition fromdegrading, because fucans are prone to degradation in acidicenvironments. In other embodiments, the method can be carried out bymaintaining the reaction mixture at or near a pH of 7 or more.

In some embodiments, the starting fucan composition may be provided as asolution. Example fucans suitable for treatment by the above methodinclude without limitation fucoidan, and the concentration of the fucanin solution can be between 0.01% w/v and 50% w/v. Impurities that can beremoved by the above method include without limitation particulates,lipids, fatty acids, phlorotannins, laminarins, alginates, proteins,Maillard reaction products, fucoxanthin, chlorophyll, bacteria, cellularcomponents and DNA.

Solid Phase Extraction

Fucans in a starting fucan composition such as a feedstock fucancomposition containing undesirable levels of impurities, including veryhigh levels of impurities such as in a raw feedstock composition, issubjected to a solid phase extraction. The methods can comprise:providing in solid form a starting fucan composition comprisingimpurities and an extraction media incapable of dissolving fucans,configured for dissolving the impurities; mixing the starting fucancomposition with the extraction media to form a mixture of anundissolved solid fucan composition and an extraction media, theextraction media containing dissolved impurities; separating thepurified undissolved solid state fucan from the extraction mediacontaining dissolved impurities; and collecting the purified/modifiedfucan as a solid after removing the purified/modified fucan from theextraction media. The separating may comprise one or more of, forexample, centrifugation, filtration, sedimentation and hydrodynamicfluid separation.

The extraction media can comprise, for example, one or more of a base, adetergent and an oxidizing agent. Suitable extraction media that do notdissolve the fucan include organic solvents with a relative polarityless than 0.765, for example, ethanol, isopropanol, methanol, benzene,diethyl ether, decamethylcyclo-pentasiloxane, ethyl acetate, butanol,hexane, heptane, heptanol, octanol and decanol. Suitable bases includewithout limitation sodium hydroxide, potassium hydroxide, lithiumhydroxide, and calcium hydroxide. Suitable oxidizing agents includewithout limitation one or more of hydrogen peroxide, urea peroxide, andoxidizing bleaches, including sodium hypochlorite. Suitable detergentsinclude without limitation nonionic surfactants, for example the Tween®,Brij® and Triton® ranges of detergents; anionic surfactants, for examplesodium dodecyl sulfate (SDS), sodium deoxycholate; and cationicsurfactants, for example benzalkomium chloride (BAC). Particular fucanslending themselves to the methods herein include, but are not limited tofucoidan. The mixing of the original, e.g., starting, fucan compositionwith the extraction media may extend from one minute to 120 hours.

The methods may further comprise desalting the starting fucancomposition before providing in solid form the starting fucancomposition. The desalting may comprise diafiltrating the starting fucancomposition as a solution in water across a molecular weight cutoff(MWCO) tangential flow filtration (TFF) filter. The diafiltrating maycomprise diafiltrating the starting fucan composition with distilledwater. The molecular weight cutoff TFF filter can have a molecularweight cutoff smaller than a desired molecular weight separation pointor target in or for the purified/modified fucan, for example a 50 kDa,70 kDa, 100 kDa, 200 kDa, 300 kDa, 500 kDa or 1000 kDa molecular weightcut-off. The diafiltrating may further comprise pre-filtering thestarting fucan composition in through a suitable pre-filter to removeparticulate matter. The method may further comprise lyophilizing asuitable starting fucan composition in solution prior to providing insolid form the starting fucan composition. The method may furthercomprise precipitating from a solution a suitable starting fucancomposition prior to providing in solid form the starting fucancomposition. Suitable precipitants include without limitation ethanol,isopropanol, propanol, acetone, methanol, dimethyl sulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran, acetonitrile, glyme,diglyme, dioxane, the solubility of the fucan decreasing as the polarityof the precipitating fluid decreases. Impurities that can be removed bythe above method include without limitation particulates, lipids, fattyacids, phlorotannins, laminarins, alginates, proteins, Maillard reactionproducts, fucoxanthin, chlorophyll, bacteria, cellular components andDNA.

Chemically Induced Precipitation

A starting fucan composition, such as a feedstock fucan composition,containing high levels of impurities including for example, suspendedparticulates, undergoes a chemically-induced precipitation ofimpurities. In certain embodiments, the methods can comprise: providinga starting fucan composition in a starting solution; precipitating theimpurities from the starting solution by means of an ionic-multivalentimpurity precipitant to provide a mixture of suspended impurities,precipitated impurities and supernatant; separating the suspendedimpurities and precipitated impurities from the supernatant solution;and collecting the supernatant solution comprising the desiredpurified/modified fucan after separating the suspended impurities andprecipitated impurities from the supernatant.

Suitable impurity precipitants include ionic-multivalent salts and/orbases of divalent and trivalent cations. Examples of such suitable saltsinclude without limitation chlorides, bromides, iodides, fluorides,sulfates, sulfites, carbonates, bicarbonates, phosphates, nitrates,nitrites, acetates, citrates, silicates and/or cyanides of alkalineearth metals, zinc, aluminum, copper and iron. Examples of such suitablebases include without limitation hydroxides and/or oxides of alkalineearth metals, zinc, aluminum, copper and/or iron. Separating thesuspended impurities and precipitated impurities from the supernatantsolution may comprise flocculating the impurities in the mixture.Suitable flocculants include without limitation potassium aluminumsulfate; sodium aluminum sulfate; ammonium aluminum sulfate; calciumchloride; sodium phosphate; aluminum hydroxide; aluminum chloride;ferric chloride; ferric sulfate; ferrous sulfate; sodium silicate;calcium silicate; calcium phosphate; zinc chloride; calcium carbonate;calcium bicarbonate; potassium sulfate; magnesium phosphate;acrylamides; acrylic acid; aluminum chlorohydate; polyaluminiumchloride; tannins; formaldehyde; melamine; N,N-dimethylaminoethylacrylate methyl chloride; N,N-dimethylaminoethyl methacrylate methylchloride quaternary; and polydiallyldimethyl-ammonium chloride. As canbe seen from the foregoing list of flocculants, in some embodiments, theflocculant can be the impurity precipitant. Separating the precipitated,suspended and/or flocculated impurities from the supernatant solutionmay comprise at least one of centrifuging, filtering, sedimentation andhydrodynamic flow separation of the mixture of impurities and thesupernatant solution.

The methods may further comprise desalting the starting fucancomposition before providing the starting fucan composition. Thedesalting may comprise diafiltrating the starting fucan composition asan aqueous solution across a TFF filter. The diafiltrating may comprisediafiltrating the starting fucan composition with distilled water. Thediafiltrating may comprise diafiltrating the starting fucan compositionacross a TFF filter with a MWCO of 5 kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDaor 100 kDa. The diafiltrating may further comprise pre-filtering thestarting fucan composition in through a suitable pre-filter to removeparticulate matter.

The methods may further comprise maintaining a pH of between about 7 and14 to inhibit or prevent degradation of fucans in acidic environments.Maintaining the pH between about 7 and 14 may comprise the addition of asuitable base, for example, sodium hydroxide. A suitable base may beadded to the starting fucan composition before precipitating impuritiesfrom the solution by means of an ionic-multivalent impurity precipitant.In other embodiments, a suitable base may be added to the mixture ofprecipitated impurities and supernatant solution after precipitatingimpurities from the solution by means of an ionic-multivalent impurityprecipitant. In yet other embodiments, a suitable base may be added tothe supernatant solution after separating the suspended impurities andprecipitated impurities from the supernatant solution.

Example fucans suitable for treatment by the above method includewithout limitation fucoidan, and the concentration of the fucan insolution can be between 0.01% w/v and 50% w/v. Impurities that can beremoved by the above method include without limitation particulates,lipids, fatty acids, phlorotannins, laminarins, alginates, proteins,Maillard reaction products, fucoxanthin, chlorophyll, bacteria, cellularcomponents and DNA.

Lysis and Flocculation

A starting fucan composition, such as a feedstock fucan composition,containing high levels of impurities undergoes lysis and flocculation.The methods in this example can comprise: providing a starting fucancomposition; rendering the starting fucan composition alkaline; addingto the starting fucan composition a cellular disrupting agent to producea reaction mixture, the cellular disrupting agent lysing cellularcomponents in the starting fucan composition and releasing into thealkaline reaction mixture lysates comprising biomolecular components;removing from the reaction mixture the cellular disrupting agent and atleast a portion of the impurities to leave undegraded the desiredpurified fucan.

The removing of the cellular disrupting agent may comprise any one ormore of precipitation, flocculation, tangential flow filtration,micellar phase separation, ionic adsorption, and hydrophobic adsorption.The removal of impurities may comprise any one or more of precipitation,flocculation, tangential flow filtration, micellar phase separation,ionic adsorption, and hydrophobic adsorption. Any of these removalmethods or combinations of removal methods may comprise centrifuging,filtering, sedimentation or hydrodynamic flow separation of any mixtureof solid and liquid phases.

Suitable cellular disrupting agents include without limitation anionic,non-ionic and cationic detergents, for example sodium dodecyl sulfate(SDS), benzalkonium chloride, Triton X 100®, Triton X 114®, Brij®detergents, Tween® detergents, sodium deoxycholate, andalkylbenzenesulfonates.

In one embodiment of the methods, the cellular disrupting agent issodium dodecyl sulfate (SDS) and the removing of the cellular disruptingagent comprises adding a precipitant for rendering the cellulardisrupting agent insoluble in the alkaline reaction mixture and tothereby precipitate the cellular disrupting agent. In this embodiment,the removing of the cellular disrupting agent may further compriseadding a flocculant to the reaction mixture to flocculate theprecipitated cellular disrupting agent and along with it at least aportion of the impurities. The removing of the cellular disrupting agentmay further comprise centrifuging after the flocculation.

Suitable precipitants for sodium dodecyl sulfate andalkylbenzenesulfonates include without limitation potassium hydroxide,potassium chloride, calcium chloride, calcium carbonate and bariumchloride. Suitable flocculants include without limitation potassiumaluminum sulfate; sodium aluminum sulfate; ammonium aluminum sulfate;calcium chloride; sodium phosphate; aluminum hydroxide; aluminumchloride; ferric chloride; ferric sulfate; ferrous sulfate; sodiumsilicate; calcium silicate; calcium phosphate; zinc chloride; calciumcarbonate; calcium bicarbonate; potassium sulfate; magnesium phosphate;acrylamides; acrylic acid; aluminum chlorohydate; polyaluminiumchloride; tannins; formaldehyde; melamine; N,N-dimethylaminoethylacrylate methyl chloride; N,N-dimethylaminoethyl methacrylate methylchloride quaternary; and polydiallyldimethyl-ammonium chloride.

It is to be understood hereby that the cellular disrupting agent mayundergo a change in the process of precipitation. For example withoutlimitation, if the cellular disrupting agent is sodium dodecyl sulfate(SDS), the precipitant may be potassium hydroxide (KOH) and the sodiumcation may be replaced as part of the precipitation process bypotassium, the resulting potassium dodecyl sulfate being insoluble inthe reaction mixture and thereby precipitating. The dodecyl sulfatecation, which functionally is the cellular disrupting portion of theSDS, stays intact in this process.

In yet other embodiments of the methods, the cellular disrupting agentmay be one or more of sodium dodecyl sulfate (SDS) and sodiumdeoxycholate and the removing of the cellular disrupting agent comprisesanionic adsorption. The anionic adsorption may comprise adding asuitable positively charged adsorbent for a suitable amount of time,followed by the removal of the adsorbent. The anionic adsorption mayfurther comprise flowing the reaction mixture over a column or filterpacked with a suitable positively charged adsorbent at a suitable flowrate.

In yet other embodiments of the methods, the cellular disrupting agentmay be benzalkonium chloride and the removing of the cellular disruptingagent comprises cationic adsorption. The cationic adsorption maycomprise adding a suitable negatively charged adsorbent for a suitableamount of time, followed by the removal of the adsorbent. The cationicadsorption may further comprise flowing the reaction mixture over acolumn or filter packed with a suitable negatively charged adsorbent ata suitable flow rate.

In yet other embodiments of the methods, the cellular disrupting agentmay be one or more of Triton X 100®, Triton X 114®, Brij® and Tween®detergents and the removing of the cellular disrupting agent comprisesmicellar phase separation. The micellar phase separation may comprisealtering the temperature of the reaction mixture such that thetemperature of the reaction mixture exceeds the cloud point of thecellular disrupting agent. The micellar phase separation may comprisecentrifuging the reaction mixture to obtain the desired phaseseparation.

In further embodiments of methods, the cellular disrupting agent may beany one or more of sodium dodecyl sulfate (SDS), benzalkonium chloride,Triton X 100®, Triton X 114®, Brij® detergents, Tween® detergents,sodium deoxycholate, and alkylbenzenesulfonates, and the and theremoving of the cellular disrupting agent comprises one or more ofhydrophobic adsorption and a combination of dilution and tangential flowfiltration (TFF). The hydrophobic adsorption may comprise adding asuitable hydrophobic adsorbent for a suitable amount of time, followedby the removal of the adsorbent. The hydrophobic adsorption may compriseflowing the reaction mixture over a column or filter packed with asuitable hydrophobic adsorbent at a suitable flow rate. The removal bydilution and TFF may comprise diluting the reaction mixture such thatthe cellular disrupting agent falls below its critical micellarconcentration and thus can be removed by means of tangential flowfiltration over a suitable molecular weight cut-off (MWCO) TFF filterthat allows for the permeation of the cellular disrupting agent from afucan containing retentate. The removal by dilution and TFF may involvediafiltering the reaction mixture over the TFF filter with a suitablenumber of diavolumes.

The methods may further comprise adding a chelating agent to thereaction mixture to chelate free multivalent cations in the reactionmixture. The chelating agent may be added after providing the startingfucan composition and before the removing of the cellular disruptingagent. The methods may further comprise quenching oxidants in thereaction mixture. The quenching of oxidants may comprise adding anoxidant-quenching agent to the reaction mixture before or after theremoving of the cellular disrupting agent.

The methods may comprise adding a bacteriostatic agent to the reactionmixture. The bacteriostatic agent may be added after providing thestarting fucan composition and before the removing of the cellulardisrupting agent. Suitable bacteriostatic agents include withoutlimitation sodium sulfite, ethylenediaminetetraacetic acid (EDTA),benzalkonium chloride, ethanol, and thiourea.

Suitable chelating agents include without limitationethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanol,ethylene diamine, porphine and citric acid. Suitable oxidant-quenchingagents include without limitation sulfite, nitrite and phosphite salts.As is evident from the above, several of the compounds listed may havemore than one function in the methods.

Suitable hydrophobic adsorbents include without limitation activatedcarbon, diatomaceous earth, acrylic ester non-ionic resins, polystyrenenon-ionic resins, styrene-divinylbenzene (DVB) non-ionic resins.Suitable anionic adsorbents include without limitation: aminefunctionalized styrene-DVB resins, amine functionalized methacrylateresins, amine functionalized methyl methacrylate resins, aminefunctionalized butyl methacrylate resins, amine functionalized agaroseresins, amine functionalized dextran resins, amine functionalizedceramic based resins, amine functionalized silicates, and lipid removalagent (LRA).

In some embodiments, the starting fucan composition may be provided as asolution. Example fucans suitable for treatment by the above methodinclude without limitation fucoidan. The starting fucan composition mayhave a fucan concentration in solution of greater than 0.1% w/v and lessthan 30% w/v. The cellular disrupting agent may have a concentration insolution of greater than 0.1% w/v and less than 60% w/v. Impurities thatmay be removed by the above method include without limitationparticulates, lipids, fatty acids, phlorotannins, laminarins, alginates,proteins, Maillard reaction products, fucoxanthin, chlorophyll,bacteria, cellular components and DNA.

Liquid-Liquid Extraction

Fucans in a starting fucan composition such as a feedstock fucancomposition containing undesirable levels of impurities undergoesliquid-liquid extraction. The methods can comprise: providing thestarting fucan composition in an aqueous starting solution; mixing thestarting solution with an organic solvent to obtain an aqueous-organicphase mixture having an aqueous portion comprising a purified/modifiedfucan, and an organic portion comprising hydrophobic impurities;separating the aqueous portion from the organic portion; and collectingthe aqueous portion comprising the purified/modified fucan.

The methods may further comprise desalting the starting fucancomposition before mixing with the aqueous starting solution an organicsolvent. The desalting may comprise diafiltrating the starting fucancomposition as a solution in water across a molecular weight cutoff(MWCO) tangential flow filtration (TFF) filter. The diafiltrating maycomprise diafiltrating the starting fucan composition with distilledwater. The molecular weight cutoff TFF filter can have a molecularweight cutoff smaller than a desired molecular weight separation pointor target in or for the purified/modified fucan, for example a 5 kDa, 10kDa, 30 kDa, 50 kDa, 70 kDa, 100 kDa, 200 kDa, 300 kDa, 500 kDa or 1000kDa molecular weight cut-off. The diafiltrating may further comprisepre-filtering the starting fucan composition in through a suitablepre-filter to remove particulate matter.

Mixing the aqueous starting solution with an organic solvent maycomprise shaking the aqueous-organic solvent mix, stirring theaqueous-organic solvent mix, exposing the aqueous-organic solvent mix tohigh-shear, recirculating the aqueous phase into the organic phase andrecirculating the organic phase into the aqueous phase.

Separating the aqueous portion from the organic portion may comprise atleast one of least one of centrifugation, decanting, separatory funnelseparation and hydrodynamic flow separation.

Suitable organic solvents for use with this method include organicsolvents with a relative polarity less than 0.765, for example, heptane,isobutyl acetate, anisole, isopropyl acetate, 1-butanol, butyl acetate,methylisobutylketone, pentane, 1-pentanol, ethyl acetate, ethyl ether,and propyl acetate. The organic phase may contain impurities, forexample without limitation, lipids, fatty acids, phlorotannin, proteins,fucoxanthin and/or chlorophyll.

Diafiltration

Fucans in a starting fucan composition such as a feedstock fucancomposition containing undesirable levels of impurities undergoesdiafiltration. The methods can comprise: subjecting the starting fucancomposition in a starting solution to diafiltration with a chelatingagent solution across a first tangential flow filtration filter toproduce a first retentate fucan composition and a permeate solution ofchelated cationic components; and subjecting the first retentate fucancomposition to diafiltration with a secondary diafiltration solutionacross a second tangential flow filtration filter to separate residualchelating agent from the first retentate fucan composition, producing asecond retentate fucan composition comprising the desiredpurified/modified fucan. Subjecting the first retentate fucancomposition to diafiltration across a second tangential flow filtrationfilter may comprise subjecting the first retentate fucan composition todiafiltration across the first flow filtration filter. That is, the samefilter can be employed in both diafiltration processes.

Subjecting the starting fucan composition to diafiltration may comprisepre-filtering the starting fucan composition through a pre-filter toremove undesired particulate material. Subjecting the starting fucancomposition to diafiltration with a chelating agent may comprisesubjecting the starting fucan composition to diafiltration with one ofethylenediamine-tetraacetic acid (EDTA), 2,3-dimercapto-1-propanol,ethylene diamine, porphine or citric acid.

The starting fucan composition may have a fucan concentration insolution of greater than 0.1% w/v and less than 30% w/v. The chelatingagent may have a concentration in solution of greater than 0.1% w/v andless than 60% w/v. The resulting first and/or second retentatecompositions can comprise a cationic content consisting essentially ofsodium and/or potassium.

FIG. 1 shows a schematic diagram of a cationic content modificationsystem 1200 for obtaining a modification of the cationic content and/orlevel of a starting fucan composition. A starting fucan composition insolution is supplied via input supply line 1202 to fucan container 1216.The starting fucoidan in a suitable solvent can be pre-filtered throughpre-filter 1204 to remove any undesired particulate matter. The gauge ofthe pre-filter will typically be greater than the largest polymermolecules to be separated by means of the cationic content modificationsystem 1200.

TFF input pump 1214 pumps starting fucan composition to TFF filter 1210via TFF supply line 1212. TFF filter 1210 is typically supplied as acassette designed to allow an input fluid supplied to it to pass overits filter on its retentate side, while allowing a permeate to exit viaone output line and treated input fluid to leave as retentate viaanother output line. TFF input pump 1214 provides a level of pressureover TFF filter 1210 between its retentate and permeate sides. In FIG. 1, the retentate of TFF filter 1210 is returned to fucan container 1216via TFF retentate return line 1218 and TFF retentate valve 1217, whilepermeate is produced via TFF permeate output line 1219 for use outsidecationic content modification system 1200 or to be discarded.

While TFF input pump 1214 recirculates the prefiltered fucoidan andretentate over TFF filter 1210, a chelating agent, for example withoutlimitation one of ethylenediamine-tetraacetic acid (EDTA),2,3-dimercapto-1-propanol, ethylene diamine, porphine or citric acid,can be added to the starting fucan composition in fucan container 1216from first diafiltration solution container 1220 via first diafiltrationsolution supply line 1225. The chelating agent is used both to replenishsolvent lost via the permeate on TFF permeate output line 1219 and/or toensure that a predetermined number of diavolumes of input fucan andchelating agent are circulated over the TFF filter 1210. The chelatingagent sequesters the cations in the starting fucan composition, inparticular multivalent cations, as chelates which then pass through theTFF filter 1210 into the permeate. By controlling first diafiltrationsolution valve 1224, the chelating agent can be added in a pulseprocess. In other embodiments, the chelating agent can be added in acontinuous mode. The number of diavolumes of chelating agent to processover TFF filter 1210 can be predetermined. The process can be continuedfor a predetermined period of time, for example between about 1 andabout 6 hours, between about 3 and about 12 hours and between about 10and about 24 hours. The process can be continued for a predeterminednumber of diavolumes of the chelating agent, for example between about 1and about 4 diavolumes, between about 3 and about 6 diavolumes, betweenabout 5 and about 10 diavolumes and between about 7 and about 20diavolumes. The process can be continued and the cationic content in thefucan container 1216 can be measured and the TFF process terminated whena desired cationic content has been attained, for example a cationiccontent comprising of below 10 parts per million (ppm), below 1 ppm,below 0.1 ppm and below 0.01 ppm of multivalent cations. Thediafiltration of the starting fucan composition in solution across TFFfilter 1210 with the first diafiltration solution affords a firstretentate fucan composition with a modified cationic content.

The next step in the process is to remove remaining chelating agent fromthe first retentate fucan composition in fucan container 1216. This canbe done by shutting first diafiltration solution valve 1224, cationiccontent modification system output valve 1206, and allowing a secondarydiafiltration solution from second diafiltration solution container 1230to enter fucan container 1216 via second diafiltration solution supplyline 1235 and second diafiltration solution valve 1234. The mix in fucancontainer 1216 is then subjected to TFF across TFF filter 1210 as beforevia TFF supply line 1212, TFF input pump 1214, TFF retentate return line1218, and TFF retentate valve 1217. The secondary diafiltration solutionmay comprise for example without limitation any one or more of deionizedwater, a solution of a bacteriostatic agent, and a salt. Thebacteriostatic agent can be, for example without limitation, sodiumsulfite, EDTA, benzalkonium chloride, ethanol, thiourea. Suitable saltsinclude without limitation sodium chloride, potassium chloride, sodiumphosphate, ammonium bicarbonate, phosphate buffered saline.

The secondary diafiltration solution is used both to replenish solventlost via the permeate on TFF permeate output line 1219 and/or to ensurethat a predetermined number of diavolumes of the first retentate fucancomposition and secondary diafiltration solution are circulated over theTFF filter 1210. By controlling second diafiltration solution valve1234, the secondary diafiltration solution can be added in a pulseprocess. In other embodiments, the secondary diafiltration solution canbe added in a continuous mode. The number of diavolumes of secondarydiafiltration solution to process over TFF filter 1210 can bepredetermined. The process can be continued for a predetermined periodof time, for example between about 1 and about 6 hours, between about 3and about 12 hours and between about 10 and about 24 hours. The processcan be continued for a predetermined number of diavolumes of thechelating agent, for example between about 1 and about 4 diavolumes,between about 3 and about 6 diavolumes, between about 5 and about 10diavolumes and between about 7 and about 20 diavolumes. for example acationic content of below 10 parts per million (ppm), below 1 ppm, below0.1 ppm and below 0.01 ppm multivalent cations. The process can becontinued and the residual chelating agent concentration in the fucancontainer 1216 can be measured and the TFF process terminated when asuitably low residual chelating agent concentration has been attained,for example a residual chelating agent level of below 10 ppm, below 1ppm, below 0.1 ppm and below 0.01 ppm. The resulting second retentatefucan composition in fucan container 1216 comprises thepurified/modified fucan product of the process of cationic contentmodification system 1200. As desired, the resulting second retentatefucan composition in fucan container 1216 can be removed from fucancontainer 1216 via cationic content modification system output line1208.

Super Critical Fluid Extraction

Fucans in a fucan composition such as a starting fucan compositioncontaining undesirable levels of impurities undergoes a supercriticalfluid extraction. The methods can comprise: providing the starting fucancomposition as a solid; placing the starting fucan composition in asupercritical extractor; subjecting the starting fucan composition inthe supercritical extractor to a suitable pressure above 70 bar; heatingthe starting fucan composition in the supercritical extractor to asuitable temperature above 30° C.; filling the supercritical extractorwith a supercritical fluid to produce a purified/modified fucan and asupercritical fluid containing extracted impurities; removing thesupercritical fluid containing extracted impurities from thesupercritical extractor after a predetermined amount of time; andrecovering the purified/modified fucan.

Filling the supercritical extractor with a supercritical fluid maycomprise filling the supercritical extractor with carbon dioxide. Thesupercritical carbon dioxide can be supplemented with between 2% v/v and10% v/v ethanol. In some embodiments, the supercritical carbon dioxidecan be supplemented with approximately 5% v/v ethanol as a co-solvent.Alternative supercritical fluids to carbon dioxide for use with thismethod include but are not limited to ethanol, ethane, hydrochloricacid, hydrofluoric acid, sulfuric acid and nitric acid.

Subjecting the starting fucan to a suitable pressure may comprisesubjecting the starting fucan composition to a pressure between about 70bar and about 2000 bar. Subjecting the starting fucan composition to asuitable temperature may comprise subjecting the starting fucancomposition to a temperature between about 30° C. and about 300° C.

Removing the supercritical fluid containing the extracted impuritiesafter a predetermined amount of time may comprise removing thesupercritical fluid after between about 5 minutes to about 50 hours, forexample between about 10 minutes to about 1 hour, between about 30minutes to about 5 hours, between about 1 hour to about 24 hours andbetween about 5 hours to about 48 hours.

The method may further comprise desalting the starting fucan compositionbefore placing the starting fucan composition in a supercriticalextractor. The desalting may comprise diafiltrating the starting fucancomposition as a solution in water across a molecular weight cutoff(MWCO) tangential flow filtration (TFF) filter. The diafiltrating maycomprise diafiltrating the starting fucan composition with distilledwater. The molecular weight cutoff TFF filter can have a molecularweight cutoff smaller than a desired molecular weight separation pointor target in or for the purified/modified fucan, for example a 50 kDa,70 kDa, 100 kDa, 200 kDa, 300 kDa, 500 kDa or 1000 kDa molecular weightcut-off. The diafiltrating may further comprise pre-filtering thestarting fucan composition in through a suitable pre-filter to removeparticulate matter.

Chemical Structural Modification

The methods, systems etc. discussed herein can comprise chemicalstructural modification of the fucans, including the fucans in a fucancomposition. The chemical structural modification may involve removal offunctional groups from the fucan, for example, 0-acetyl, N-acetyl,methoxy, hydroxyl, carboxylic and/or sulfate functional groups from thefucan structure. The chemical structural modification may involve theuse of a wide variety of chemical reagents, for example, acids, bases,detergents and/or oxidizing agents.

Tangential Flow Filtration

Some of the methods discussed herein utilize tangential flow filtration(TFF). Consistent with typical identification of tangential flowfiltration (TFF) filters, the nominal molecular weight cut-off (MWCO)value for a given TFF filter will selectively retain on its retentateside a solution containing molecules that did not cross the filterbarrier and thus generally have molecular weights and/or sizes greaterthan the molecular weight of molecules that do cross/permeate thebarrier to the permeate side. Thus, molecular weight cut-off values forTFF filters are typically not absolute for any given polymer or nominalcut-off value: a given TFF filter will pass or retain some moleculesboth above and below the nominal molecular weight cut-off. The actualcut-off/selectively values and effects of a nominal TFF filter for aparticular polymer can be routinely determined for the particularpolymer.

A number of factors can affect the permeation behavior of the TFFfilters. These factors may be dependent on the TFF filters themselves ordependent on an attribute of the target polymers, for example thefolding behavior and folded structure of the target polymer can affectthe behavior of the target polymer in crossing/not-crossing the TFFfilter's MWCO barrier. Regarding the TFF filters themselves, as isknown, a number of factors can affect the permeation behavior of the TFFfilters. For example, manufacturing methods can cause a variety of holesizes within the specific TFF filter, which variety can include holesboth larger and smaller than the nominal MWCO. Thus, a TFF filter havinga nominal molecular weight cut-off value will substantially pass/retainmolecules at the nominal molecular weight cut-off value, but can alsopass/retain some molecules below and/or above such value.

Gel Permeation Chromatography

Gel permeation chromatography was employed to evaluate the molecularweight distributions obtained for the experimental examples. There are alarge number of different parameters, columns and standards availablefor use in gel permeation chromatography, resulting in a variety ofinstrumentation set-ups available for the analysis of molecular weight.For molecular weight determinations herein, the GPC were conducted usingthe following parameters: The mobile phase was 0.1M sodium nitrate runat 0.6 mL/min. The column compartment and detector were at 30° C. AWaters 2414 refractive index detector was used for detection.

Suitable GPC columns include GPC columns compatible with aqueoussolvents, for example columns packed with at least one of sulfonatedstyrene-divinylbenzene, NH-functionalized acrylate copolymer network,modified silica and hydroxylated polymethacrylate-based gel. For theanalyses herein, three columns were used in series, comprising one 40 mmlong guard column with an inner diameter (ID) of 6 mm packed with 6 μmparticle size hydroxylated polymethacrylate-based gel, followed by afirst 300 mm analytical GPC column with a 7.8 mm ID packed with 12 μmparticle size hydroxylated polymethacrylate-based gel that has anexclusion limit of about 7,000 kDa and an effective molecular weightrange of between about 50 kDa and about 5,000 kDa, followed by a second300 mm analytical GPC column with a 7.8 mm ID packed with 10 μm particlesize hydroxylated polymethacrylate-based gel that has an exclusion limitof about 7,000 kDa and an effective molecular weight range of betweenabout 1 kDa and about 6,000 kDa. The total effective molecular weightrange of the column set up was between about 1 kDa and about 6,000 kDa.An example of this column set up can be Ultrahydrogel®guard-Ultrahydrogel® 2000-Ultrahydrogel® Linear columns connected inseries.

Samples run were quantified against a standard curve comprising oftraceable standards from the American Polymer Standards Corporation:DXT3755K (peak molecular weight=2164 kDa), DXT820K (peak molecularweight=745 kDa), DXT760K (peak molecular weight=621 kDa), DXT670K (peakmolecular weight=401 kDa), DXT530K (peak molecular weight=490 kDa),DXT500K (peak molecular weight=390 kDa), DXT270K (peak molecularweight=196 kDa), DXT225K (peak molecular weight=213 kDa), DXT150K (peakmolecular weight=124 kDa), DXT55K (peak molecular weight=50 kDa), DXT50K(peak molecular weight=44 kDa) and DXT5K (peak molecular weight=4 kDa),the peak molecular weights of these standards being between about 4 kDaand about 2,200 kDa. The standard curve used may, for example, includeDextran 3755 kDa, at least one of Dextran 50 kDa and Dextran 55 kDa, andbetween 3 to 6 additional traceable standards discussed herein, thecalibration points being the peak molecular weights of the calibrantsused. An example calibration curve may consist of DXT3755K, DXT 820K,DXT530K, DXT500K, DXT225K and DXT55K. The columns used herein had atotal effective molecular weight range that encompassed and extendedbeyond the peak molecular weight range of the standards used forquantification of the fucans.

A molecular weight stated for a fucan/fucoidan polymer herein is a valueof molecular weight about which there will always be a distribution ofmolecules of higher and lower molecular weights, increasing ordecreasing in amount or percentage as the molecular weight increases ordecreases away from the specified molecular weight. The distributionmay, but is not required to, have a generally Gaussian or distortedGaussian shape.

Results in the tables herein contain abbreviations used for certaincharacteristics of a molecular weight distribution. Gel permeationchromatography is denoted by GPC, peak retention time is denoted by PRT,peak molecular weight is denoted by PMW, weight average molecular weightis denoted by WAMW, number average molecular weight is denoted by NAMW,percentage distribution is denoted by % dist., molecular weight isdenoted by MW, polydispersity index is denoted by PDI and molecularweight cutoff is denoted by MWCO.

Diseases and Conditions

Fibrous Adhesions

A fibrous adhesion is a type of scar that forms between two parts of thebody, usually after surgery (surgical adhesion). Fibrous adhesions cancause severe problems. For example, fibrous adhesions involving thefemale reproductive organs (ovaries, Fallopian tubes) can causeinfertility, dyspareunia and severe pelvic pain. Fibrous adhesions thatoccur in the bowel can cause bowel obstruction or blockage, and fibrousadhesions can also form in other places such as around the heart, spineand in the hand. In addition to surgery, fibrous adhesions can be causedfor example by endometriosis, infection, chemotherapy, radiation, traumaand cancer.

A variety of fibrous adhesions are discussed in this document. Termssuch as surgical adhesions, post-surgical adhesions, postoperativeadhesions, adhesions due to pelvic inflammatory disease, adhesions dueto mechanical injury, adhesions due to radiation, adhesions due toradiation treatment, adhesions due to trauma, and adhesions due topresence of foreign material all refer to adherence of tissues to eachother due to a similar mechanism and are all included in the termfibrous adhesions.

Fibrous adhesion formation is a complex process in which tissues thatare normally separated in the body grow into each other. Surgicaladhesions (also known as post-surgical adhesions) develop from theotherwise normal wound healing response of the tissues to trauma andhave been reported to occur in over two-thirds of all abdominal surgicalpatients (Ellis, H., Surg. Gynecol. Obstet. 133: 497 (1971)). Theconsequences of these fibrous adhesions are varied and depend upon thesurgical site or other site, such as a disease site, involved. Problemsmay include chronic pain, obstruction of the intestines and even anincreased risk of death after cardiac surgery (diZerega, G. S., Prog.Clin. Biol. Res. 381: 1-18 (1993); diZerega, G. S., Fertil. Steril.61:219-235 (1994); Dobell, A. R., Jain, A. K., Ann. Thorac. Surg. 37:273-278 (1984)). In women of reproductive age, fibrous adhesionsinvolving the uterus, fallopian tubes or ovaries are estimated toaccount for approximately 20% of all infertility cases (Holtz, G.,Fertil. Steril. 41: 497-507 (1984); Weibel, M. A. and Majno, G. Am. J.Surg. 126: 345-353 (1973)).

The process of fibrous adhesion formation initially involves theestablishment of a fibrin framework and normal tissue repair. The normalrepair process allows for fibrinolysis alongside mesothelial repair.However, in fibrous adhesion formation the fibrin matrix matures asfibroblasts proliferate into the network and angiogenesis occursresulting in the establishment of an organized fibrous adhesion withinabout 3 to 5 days (Buckman, R. F., et al., J. Surg. Res. 21: 67-76(1976); Raferty, A. T., J. Anat. 129: 659-664 (1979)). Inflammatoryprocesses include neutrophil activation in the traumatized tissues,fibrin deposition and bonding of adjacent tissues, macrophage invasion,fibroblast proliferation into the area, collagen deposition,angiogenesis and the establishment of permanent fibrous adhesiontissues.

Various attempts have been made to prevent surgical adhesions. Theseinvolve pharmacological approaches targeted at influencing thebiochemical and cellular events that accompany surgical traumas well asbarrier methods for the separation of affected tissues. For example, theuse of peritoneal lavage, heparinized solutions, procoagulants,modification of surgical techniques such as the use of microscopic orlaparoscopic surgical techniques, the elimination of talc from surgicalgloves, the use of smaller sutures and the use of physical barriers(films, gels or solutions) aiming to minimize apposition of serosalsurfaces, have all been attempted. Currently, preventive therapies alsoinclude prevention of fibrin deposition, reduction of inflammation(steroidal and non-steroidal anti-inflammatory drugs) and removal offibrin deposits.

Interventional attempts to prevent the formation of post-surgicaladhesions have included the use of hydroflotation techniques or barrierdevices. Hydroflotation involves the instillation of large volumes ofpolymer solutions such as dextran (Adhesion Study Group, Fertil. Steril.40:612-619 (1983)), or carboxymethyl cellulose (Elkins, T. E., et al.,Fertil. Steril. 41:926-928 (1984)), into the surgical space in anattempt to keep the organs apart. Synthetic barrier membranes made fromoxidized regenerated cellulose (e.g., Interceed™),polytetrafluoroethylene (Gore-tex surgical membrane) and fullyresorbable membranes made from a modified hyaluronicacid/carboxymethylcellulose (HA/CMC) combination (Seprafilm™) have alsobeen used to reduce post-surgical adhesion formation in both animals andhumans (Burns, J. W., et al., Eur. J. Surg. Suppl. 577: 40-48 (1997);Burns, J. W., et al., Fertil. Steril. 66:814-821 (1996); Becker, J. M.,et al., J. Am. Coll. Surg. 183:297-306 (1996)). The success of theseHA/CMC membranes may derive from their ability to provide tissueseparation during the peritoneal wound repair process when fibrousadhesions form. The membranes were observed to form a clear viscouscoating on the injured tissue for 3-5 days after application, a timeperiod that is compatible with the time course of post-surgical adhesionformation (Ellis, H., Br. J. Surg. 50: 10-16 (1963)). Unfortunately,limited success has been seen with these methods.

Peritonitis involves inflammation of the peritoneum. Peritonitis cancause severe problems. For example, abdominal pain, abdominal tendernessand abdominal guarding. Peritonitis may involve spontaneous, anatomicand/or peritoneal dialysis related inflammation. Peritonitis may involvean infection, for example, perforation of a hollow viscus, disruption ofthe peritoneum, spontaneous bacterial peritonitis, and systemicinfections may result in infection and peritonitis. Peritonitis may alsonot involve an infection, for example, leakage of sterile body fluidsinto the peritoneum, and sterile abdominal surgery may result inperitonitis. Various attempts have been made to prevent and/or treatperitonitis. For example, general supportive measures such asintravenous rehydration, antibiotics, and surgery. There is an unmetneed for compounds, compositions, methods and the like (includingdelivery approaches) to inhibit, or otherwise treat and/or prevent,peritonitis, preferably more effectively with few side effects.

The purified/modified fucans discussed herein can be used to treatfibrous adhesions in a patient and can be included as a component of, orbe, a purified/modified fucan medical composition, medical device,combination or pharmaceutical product configured and can be composed totreat fibrous adhesions. For example, a purified/modified fucan medicalcomposition or medical device comprising between about 0.02 mg/mL toabout 100 mg/mL, for example 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.5 mg/mL,0.9 mg/mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL 7.5 mg/mL, of a purified/modifiedfucan herein dissolved in a physiological salt solution. Thephysiological salt solution can be, for example, Lactated Ringer'sInjection USP (LRS), normal saline and physiological Dextran solution.

The purified/modified fucan medical compositions and medical devices,which can be liquid medical compositions and devices, herein can containpharmaceutically acceptable excipients such as buffers, stabilizers,preservatives, adjuvants, etc. Such purified/modified fucan medicalcompositions and medical devices can be used to treat fibrous adhesionspre-, during, or post-surgery by administering between about 0.01 mL/kg(per kilogram bodyweight of the patient or target) to about 10 mL/kg or15 mL/kg of the fucan medical compositions or devices in the precedingparagraph. Doses and device quantities include, for example, about 0.03mL/kg, 0.1 mL/kg, 0.2 mL/kg, 0.4 mL/kg, 0.5 mL/kg, 0.6 mL/kg, 1 mL/kg,1.2 mL/kg, 2 mL/kg, 3 mL/kg, 4 mL/kg, 5 mL/kg, 8 mL/kg, 10 mL/kg and 15mL/kg of the purified/modified fucan medical composition or medicaldevice to the surgical site of the patient. In further embodiments, suchpurified/modified fucan medical compositions and medical devices can beused to treat fibrous adhesions at any selected target site, for examplelesions, abrasions, injury sites, surgical sites and post-surgical sitesby administering between about 0.04 mg/kg or 0.1 mg/kg to about 25 mg/kgor 50 mg/kg. Some examples of such doses include, for example, about0.04 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.3mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 8 mg/kg, 10 mg/kg,15 mg/kg, 20 mg/kg, 25 mg/kg and 50 mg/kg of the fucans herein,including for example the purified/modified fucans herein, to thesurgical site of the patient. The administering can be accomplished, forexample, by instilling a liquid medical composition or medical devicegenerally throughout the target area; directing the liquid medicalcomposition or medical device at a specific location(s) within thetarget area; spraying the liquid medical composition or medical devicegenerally or at a specific location(s) within the target area; or,spraying or otherwise delivering the liquid medical composition ormedical device via an applicator, which can be a spray applicatorthrough a trocar, catheter, endoscope or other minimally invasivedevice, onto a specific location(s) that a surgeon or other practitionerhas identified as particularly susceptible to or concerning fordevelopment of fibrous adhesions. In another aspect, the administeringcan be done after opening of the surgical wound but before the surgicalprocedure; during the surgical procedure, or after the surgicalprocedure but before the surgical wound has been closed. If desired, theliquid medical composition or medical device can also be administeredafter the surgery is completed (for example through a syringe andneedle) and can be administered to non-surgical target sites as well.The surgical site of the patient can be, for example, at least one ofthe pelvic cavity, abdominal cavity, dorsal cavity, cranial cavity,spinal cavity, ventral cavity, thoracic cavity, pleural cavity,pericardial cavity, skin, joints, muscles, tendons and ligaments. Theadministering of the purified/modified fucan medical composition ormedical device into the surgical site of the patient can be accomplishedin less than about 15 minutes, 10 minutes, 8 minutes, 6 minutes, 5minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 seconds, 30seconds, 20 seconds, 15 seconds, 10 seconds and 5 seconds.

Examples of administering the purified/modified fucan medicalcomposition or medical device to a surgical site include withoutlimitation administering the purified/modified fucan medical compositionor medical device at the surgical site of a Cesarean section surgicalprocedure; a microvascular free flap reconstruction surgical procedure,a full thickness skin graft surgical procedure, a V-Y advancement flapsurgical procedure, a fasciocutaneous rotation flap surgical procedure,an arthroplasty surgical procedure, a mastectomy surgical procedure, asequestrectomy surgical procedure, a saucerization surgical procedure,an osteotomy surgical procedure, an osteoplasty surgical procedure, apatellectomy surgical procedure, a synovectomy surgical procedure, acapsulectomy surgical procedure, a tendon or ligament repair surgicalprocedure, a tenolysis surgical procedure, a tenotomy surgical, afasciotomy surgical procedure, a meniscal repair surgical procedure, avertebrectomy surgical procedure, a ethmoidectomy surgical procedure, aCaldwell Luc's operation surgical procedure, a dacryocystorhinostomysurgical procedure, a lysis nasal synechia surgical procedure, athymectomy surgical procedure, a pneumonolysis surgical procedure, apneumonectomy surgical procedure, thoracoplasty surgical procedure, abilobectomy surgical procedure, a portal hypertension surgery surgicalprocedure, a splenectomy surgical procedure, a esophagectomy surgicalprocedure, a peritonitis surgery surgical procedure, a gastrectomysurgery surgical procedure, a jejunojejunostomy surgery surgicalprocedure, a laparoscopic cholecystectomy surgery surgical procedure, alaparoscopic common bile duct exploration surgical procedure, agastroenterostomy surgical procedure, a bariatric surgery surgicalprocedure, a bowel resection & anastomosis surgical procedure, asegemental hepatectomy surgical procedure, a lobectomy surgicalprocedure, a pancreatomy surgical procedure, a pancreaticoduodenectomysurgical procedure, a tumor resection surgical procedure, a laparoscopicnephrectomy surgical procedure, a cystectomy surgical procedure, anabdominal or pelvic adhesion lysis surgical procedure, ahysterosalpingostomy surgical procedure, a salpingoplasty surgicalprocedure, an ectopic pregnancy laparoscopic surgery surgical procedure,a joint replacement surgery surgical procedure, a broken bone repairsurgical procedure, a hysterectomy surgical procedure, a gallbladderremoval surgical procedure, a heart bypass surgical procedure, anangioplasty surgical procedure, an atherectomy surgical procedure, abreast biopsy surgical procedure, a carotid endarterectomy surgicalprocedure, a cataract surgery surgical procedure, a coronary arterybypass surgical procedure, a dilation and curettage surgical procedure,a hernia repair surgical procedure, a lower back pain surgery surgicalprocedure, a partial colectomy surgical procedure, prostatectomysurgical procedure and a tonsillectomy surgical procedure, after openingthe surgical wound, during surgery, before closing the surgical woundand/or after closing the surgical wound.

Cancers Generally

Cancer has been the second leading cause of death in the U.S. andaccounts for over 20% of all mortalities. Cancer is a proliferativedisease and is characterized by the uncontrolled division of certaincells, which may lead to the formation of one or more tumors. A numberof methods are used to treat cancer, including surgery, radiation,chemotherapy and combinations thereof. Although surgery is a relativelycommon method used for some localized tumors, there is still asignificant chance of tumor recurrence after tumor excision.

Treating cancers and other proliferative diseases has been limited bythe potential for damage or toxicity to non-cancerous, healthy tissues.In radiation and surgical treatments, the procedure has been generallyconfined to and proximal to the tumor sites. However, there can besignificant risk to patients undergoing surgical removal of canceroustissues (e.g., in removal of prostate or brain tumors there can be asignificant risk of non-repairable damage to surrounding vital tissues,for example via potential reduced need for resection of non-tumortissues. Furthermore, in focused radiation treatment, which has beengiven as a first line treatment for prostate cancer, there are similarrisks. In the chemotherapeutic treatment of cancer, the drug has beenadministered systemically, so that the whole body is exposed to thedrug. These drugs are designed to be toxic to cancer cells, but they arealso (generally) toxic to non-cancerous cells so that patients becomequite ill when undergoing drug treatments for cancer. Throughexperience, oncologists are able to give doses of these drugs that maybe tolerated by some patients. However, these doses are often notsuccessful in treating cancers.

One problem with any method of treating cancer has been the localrecurrence of the disease. For example, approximately 700,000 Americansare diagnosed with localized cancer annually (approximately 64% of allcancer patients) and almost half a million are treated using surgicalmethods. Unfortunately, 32% of patients treated with surgery relapseafter the initial treatment (approximately 21% relapse at the initialsurgical site and 11% at distant metastatic sites). Almost 100,000patients die annually due to localized recurrence of cancer. This hasbeen especially true in breast cancer where 39% of patients undergoinglumpectomy will experience local recurrence of the disease.

Staging is a method of judging the progress of the cancer (solid tumor)in a patient. A simplified approach puts patients into three groups orstages based on how far the cancer has advanced:

Stage 1: The cancer can be treated by surgically removing part of theorgan. This is also known as the resectable stage.

Stage 2: The cancer has advanced past the point of being resectable butis still confined to the organ itself.

Stage 3: The tumor has spread to other organs.

Many cancers are treated with anti-proliferative agents including, forexample, 5-fluorouracil (Efudex®), vinca alkaloids (for example,vincristine (Oncovin®)), anthracyclines (for example, doxorubicin(Adriamycin®)), cisplatin (Platinol-AQ®), gemcitabine hydrochloride(Gemzar®), methotrexate and paclitaxel. Some examples of the toxicitiesassociated with the anti-proliferative agents, methotrexate andpaclitaxel, are discussed elsewhere herein. Methotrexate has been usedto treat several cancers including, for example, bladder, breast,cervical, head and neck, hepatic, lung, and testicular cancers.Paclitaxel has been used to treat several cancers including, forexample, ovarian, breast, and non-small cell lung cancers (Compendium ofPharmaceutical and Specialties Thirty-fifth Edition, 2000).

Toxicities due to 5-fluorouracil can include cardiovascular toxicitysuch as myocardial ischemia; central nervous system toxicities such aseuphoria, acute cerebellar syndrome and ataxia; dermatologic toxicitiessuch as alopecia and dermatitis; gastrointestinal toxicities such asnausea, vomiting and oral or gastrointestinal ulceration; hematologictoxicities such as leukopenia, thrombocytopenia and anemia;hypersensitivity toxicities such as anaphylaxis and contacthypersensitivity; ocular toxicities such as increased lacrimation,photophobia and conjunctivitis; and, other toxicities such as fever.5-fluorouracil has been used to treat many cancers including, forexample, breast, colorectal, gastric, hepatic, bladder, head and neck,non-small cell lung, ovarian, pancreatic, and prostate cancers(Compendium of Pharmaceutical and Specialties Thirty-fifth Edition,2000).

Toxicities due to vincristine include central nervous system toxicitiessuch as seizures in children and hallucinations; dermatologic toxicitysuch as alopecia; extravasation toxicity such as vesicant;gastrointestinal toxicities such as nausea, vomiting, constipation andstomatitis; hematologic toxicity such as myelosuppression; neurologictoxicities such as peripheral neuropathy and autonomic neuropathy;ocular toxicities such as double vision, transient blindness and opticatrophy; renal/metabolic toxicities such as urinary retention,hyperuricemia and bladder atony; respiratory toxicity such as shortnessof breath; and, other toxicity such as fever in children. Thisanti-proliferative agent has been used to treat several cancersincluding, for example, Hodgkin's disease, small cell lung, Wilm'stumor, and testicular cancers (Compendium of Pharmaceutical andSpecialties Thirty-fifth Edition, 2000).

Toxicities due to doxorubicin include cardiovascular toxicities such aselectrocardiographic abnormalities and cardiomyopathy; dermatologictoxicities such as alopecia and nail changes; extravasation hazardtoxicity such as vesicant; gastrointestinal toxicities such and nausea,vomiting and stomatitis; genitourinary toxicity such as red colorationof urine; hematologic toxicity such as myelosuppression;hypersensitivity toxicities such as anaphylaxis and skin rash; oculartoxicity such as conjunctivitis; reproductive toxicity such asinfertility; and, other toxicity such as hyperuricemia. Thisanti-proliferative agent has been used to treat several cancersincluding, for example, breast, small cell lung, and ovarian cancers(Compendium of Pharmaceutical and Specialties Thirty-fifth Edition,2000).

Toxicities due to cisplatin include cardiovascular toxicity such aselectrocardiographic changes; dermatologic toxicity such ashyperpigmentation; extravasation hazard toxicity such as irritant;gastrointestinal toxicities such as nausea and vomiting; hematologictoxicities such as myelosuppression and hemolytic anemia;hypersensitivity toxicity such as anaphylactic; neuromuscular toxicitysuch as peripheral neuropathy and acute encephalopathy; ocular toxicitysuch as retrobulbar neuritis; otologic toxicities such as hearing lossand tinnitus; renal/metabolic toxicities such as toxic nephropathy andhypokalemia; and, other toxicity such as infertility. Thisanti-proliferative agent has been used to treat several cancersincluding, for example, bladder, small cell lung, ovarian, testicular,brain, breast, cervical, head and neck, hepatoblastoma, and thyroidcancers (Compendium of Pharmaceutical and Specialties Thirty-fifthEdition, 2000). Toxicities due to gemcitabine hydrochloride include, forexample, hematologic toxicities such as myelosuppression;gastrointestinal toxicities such as nausea, vomiting and stomatitis;hepatic toxicities such as transient elevations of serum transaminases;renal toxicities such as proteinuria, hematuria, hemolytic uremicsyndrome and renal failure; dermatologic toxicity such as rash andalopecia; edema toxicities such as edema and peripheral edema; and,other toxicity such as fever. This anti-proliferative agent has beenused to treat pancreatic and non-small cell lung cancers (Compendium ofPharmaceutical and Specialties Thirty-fifth Edition, 2000).

The present discussion comprises prevention or treatment of localizedcancers or solid tumors that can be treated include those of theprostate, breast, pancreas, liver, kidney, genitourinary system, brain,gastrointestinal system, respiratory system, and head and neck. Thecompositions, etc., herein may prevent or treat cancers, includingmetastases, by allowing controlled release of purified/modified fucan ata site somewhat distant from the target tumors by allowing effectiveconcentrations of the purified/modified fucan to reach the tumors and/ormetastases by diffusion or even systemic transport. Some of thesecancers are discussed further in the following paragraphs.

Prostate Cancer

Prostate cancer is a malignant tumor that arises in the cells lining theprostate gland. In the U.S., an estimated 200,000 patients will developprostate cancer this year, and more than 30,000 will die of the disease.Prostate cancer has a death to new cases ratio of ˜15%. The cancer mayremain within the prostate, or it may spread to surrounding tissues orto distant sites (most often lymph nodes and bone). Usually prostatecancer spreads silently, producing symptoms only when it has progressedbeyond the prostate. If prostate cancer has been diagnosed and treatedduring early stages, in some studies patients have had a 5-year survivalrate of 94%.

Prostate cancer is often discussed as a disease of men over age 50. Infact, 80% of men with prostate cancer are 60 years of age and older. Aman's chances of being diagnosed with prostate cancer during hislifetime are about 1 in 10, roughly the same as a woman's chances ofhaving breast cancer. The number of reported new cases has risendramatically in recent years as a result of improved tests that candetect the disease early in its development, often long before symptomsappear. The likelihood of developing prostate cancer in any given yearincreases with age but rises dramatically after age 50.

Current treatment options for prostate cancer depend upon the extent ofdisease progression, the patient's age and overall health. Elderlypatients, who have only early stage cancer or who suffer fromadditional, more serious diseases, may be treated conservatively,whereas those whose cancer is advanced may undergo more aggressivetreatment. Prostate cancer has been treated by various methods,including radiation therapy (external beam radiation or brachytherapy),hormone withdrawal or castration (surgical or chemical),anti-proliferative agents, surgery, and expectant therapy (that is,“watchful waiting”). No treatment guarantees an absolute cure, and somehave considerable side effects.

Early stage prostate cancer (that is, the tumor is localized to theprostate) may be treated with “watchful waiting”. Surgery for prostatecancer has been recommended for patients whose overall health has beenotherwise good and the tumor is confined to the prostate gland. A commontreatment for localized cancer of the prostate in men under the age of70 has been radical prostatectomy (that is, surgical removal of theprostate).

Patients whose cancer is localized in the prostate area are commonlytreated with external beam radiation (EBR). The radiation kills cancercells and shrinks tumors. EBR accounts for less than 20% of localizedprostate cancer treatment, with approximately 50% of these patientsexperiencing post radiation recurrences of the disease. Combined withearly stage prostate cancer detection and increased demand frompatients, brachytherapy (i.e., local radiation therapy) use has beenexpected to grow. In 1995, only 2.5% of newly diagnosed patients weretreated using brachytherapy. Brachytherapy involves the implantation ofradioactive metal “seeds” in the prostate tumor.

Treatment for prostate cancer that has spread involves removal of thetesticles or hormone therapy. Both are used to inhibit or stop theproduction of the testosterone that has been driving the cancer growth.Approximately 20% of all prostate cancer patients undergo hormonewithdrawal therapy. Hormone therapies include goserelin acetate(Zoladex®) or leuprolide acetate (Lupron®). Anti-proliferative agentsused to treat prostate cancer have included 5-fluorouracil.

Breast Cancer

In the U.S., breast cancer has been the most common cancer among women,with about 180,000 new cases diagnosed every year (male breast canceraccounts for about 5% of all diagnosed breast cancers). It has beensurpassed only by lung cancer as a cause of death in women, and it hasbeen responsible for approximately 50,000 deaths annually. An Americanwoman has a one in eight (or about 13%) chance of developing breastcancer during her lifetime. Over the past decade, most reported breastcancers were small, primary (arising independently; not caused by ametastasis) tumors. Roughly 70% to 80% of newly diagnosed patientsexhibited early-stage disease (Stage 1 or 2), and a majority had noinvolvement of the axillary (underarm) lymph nodes.

Most breast cancers are carcinomas (that is, malignant tumors that growout of epithelial tissues). Less than 1% of breast cancers are sarcomas,or tumors arising from connective tissue, bone, muscle or fat. Inaddition, most breast cancers (about 75%) are ductal carcinomas, arisingin the tissues that line the milk ducts. A much smaller number ofcancers (about 7%) are found within the breast lobules and are calledlobular carcinomas. Paget's disease (cancer of the areola and nipple)and inflammatory carcinoma account for nearly all other forms of breastcancer.

Breast cancer treatment has been complicated and depends on manyfactors. Two important factors are the type of tumor and the stage ofprogression. Tumor characteristics, in particular, help to separateindividuals into two groups: (1) those who are at low risk of cancerrecurrence and (2) those who are at high risk of cancer recurrence.Specific prognostic factors place patients in either of these groups.These factors include tumor size; presence of female sex hormoneestrogen and progesterone (ER/PR) receptors; cellular growth cycle phase(whether tumor cells are actively dividing or are in “S-phase”);presence of a protein known as “her-2-neu protein”; tumor grade, anindicator of tumor cell differentiation or change; and, tumor ploidy,the number of sets of genetic material within tumor cells.

Treatment of primary disease without significant lymph node involvementhas been by lumpectomy and radiotherapy. More significant lymph nodeinvolvement may warrant mastectomy and removal of auxiliary lymph nodes.At this stage the chance of metastasis and local recurrence has beenhigh. Treatment of metastatic disease has been palliative, involvingradiation therapy and chemotherapy, which are immunosuppressive,cytotoxic and leukopenia. Anti-proliferative agents including, forexample, 5-fluorouracil, doxorubicin, methotrexate, and paclitaxel, havebeen approved for use against breast cancer.

Pancreatic Cancer

The pancreas is an organ of the digestive system located near thestomach and small intestine. It has two major functions: the productionof enzymes and hormones. Cancers of the pancreas can occur in theexocrine (i.e., enzymes) pancreas (e.g., classic pancreaticadenocarcinomas) or can occur in the endocrine (i.e., hormones)pancreas.

Cancers of the exocrine pancreas are a very serious health issue. In theU.S., approximately 28,000 patients are diagnosed with pancreaticcancer, while about the same number die annually from this disease.Pancreatic cancer occurs equally in males and females. Due todifficulties in diagnosis, the intrinsic aggressive nature of pancreaticcancers, and the sparse systemic treatment options available, onlyapproximately 4% of patients diagnosed with pancreatic adenocarcinomalive for 5 years after diagnosis. Pancreatic cancer has been the 5^(th)leading cause of cancer death, following breast, lung, colon, andprostate cancer.

The choice of treatment for pancreatic cancer depends largely on thestage of the tumor. Possible treatments include surgery,anti-proliferative agents, radiation, and biological therapy. Surgeryhas been usually reserved for Stage 1 patients whose cancer is deemedresectable. Sometimes a combination of therapies, such as radiation andanti-proliferative agent given before or after surgery, can increase apatient's chances of survival. Pancreatic cancer that is deemedunresectable (usually Stage II or later) may be treated usinganti-proliferative agents in clinical trials. Anti-proliferative agents,such as, for example, gemcitabine or 5-fluorouracil have had some effectagainst pancreatic cancer and gemcitabine has been used as a palliativeagent. Toxicities due to these anti-proliferative agents are discussedelsewhere herein. Radiation therapy has some effect against pancreaticcancer when used in combination with chemotherapy. Radiation therapyalone may subdue symptoms. This form of treatment has also been used inStage II or later pancreatic cancers.

Bladder Cancer

In 1998, it was estimated that over 54,000 new cases of bladder cancerwould be diagnosed in the U.S. and about 15,000 deaths would beattributed to the disease. Bladder cancer has been the fourth mostcommon cancer among American men and the ninth most common cancer amongAmerican women. It occurs three times more frequently in men than inwomen. Primarily a disease of older men, bladder cancer has been asignificant cause of illness and death. The risk of bladder cancerincreases steeply with age (80% of cases occur in people older than 50years), with over half of all bladder cancer deaths occurring after age70. In white men over 65, the annual disease rate of bladder cancer hasbeen approximately 2 cases per 1,000 persons; this contrasts with a rateof 0.1 cases per 1,000 persons under 65. During one's lifetime, theprobability of developing bladder cancer has been greater than 3%;however, the probability of dying, from bladder cancer has been small(<1%). Bladder cancer rarely occurs in people who are younger than 40years of age.

Recent studies suggest that certain genes and inherited metabolicabilities may play a role in bladder cancer. Transitional cell carcinoma(TCC) has been the most common form of bladder cancer. TCC usuallyoccurs as a superficial (surface), papillary (wart-like), exophytic(outward-growing) mass upon a stalk-like base. In some cases, though,TCC may be attached on a broad base or it may appear ulcerated (withinan indented lesion). Papillary TCCs often start out as areas ofhyperplasia that later dedifferentiate or lose individual cellcharacteristics. Only about 10% to 30% of papillary TCCs develop intoinvasive cancers. By contrast, nonpapillary forms of TCC are more likelyto become invasive. As noted, such TCCs may appear ulcerated or flat.Flat, nonpapillary TCC that has been made up of anaplastic epitheliumhas been classified as carcinoma in situ (CIS or TIS). The tissue of CIScontains cells that are large, have noticeable nucleoli (round bodywithin a cell; involved in protein synthesis), and lack normal polarity.

The treatment of bladder cancer depends upon many factors. The mostimportant of these factors are the type of tumor that is present and itsstage. Common treatments include transurethral resection (TUR),electrosurgery, laser surgery, intravesical therapy, anti-proliferativeagents, surgical therapy, cystectomy, and radiation therapy. Examples ofanti-proliferative agents used to treat bladder cancer include, forexample, 5-fluorouracil, cisplatin and methotrexate. Toxicities due tothe anti-proliferative agents, 5-fluorouracil, cisplatin, andmethotrexate, are discussed elsewhere herein.

Brain Cancer

Brain tumors are often inoperable and more than 80% of patients diewithin 12 months of diagnosis. Approximately 18,000 new cases of primaryintracranial (brain) cancer are diagnosed each year in the U.S. Thisrepresents about 2 percent of all adult cancers. More than 50 percent ofthese are high-grade gliomas (i.e., glioblastoma multiform andanaplastic astrocytoma tumors). Patients with these tumors often sufferfrom severe disabilities such as motor dysfunction, seizures, and visionabnormalities.

Tumors that begin in brain tissue are known as primary brain tumors.Primary brain tumors are classified by the type of tissue in which theybegin. The most common brain tumors are gliomas, which begin in theglial (supportive) tissue. Others include astrocytomas, brain stemgliomas, ependymomas and oligodendrogliomas.

Surgical removal of brain tumors has been recommended for most types andin most locations and should be as complete as possible within theconstraints of preservation of neurologic function. An exception to thisrule has been for deep-seated tumors, such as pontine gliomas, which arediagnosed on clinical evidence and are treated without initial surgeryapproximately 50% of the time. In many cases, however, diagnosis bybiopsy is performed. Stereotaxic biopsy can be used for lesions that aredifficult to reach and resect. Patients who have brain tumors that areeither infrequently curable or unresectable should be consideredcandidates for clinical trials that evaluate radiosensitizers,hyperthermia, or interstitial brachytherapy used in conjunction withexternal-beam radiation therapy to improve local control of the tumor orfor studies that evaluate new drugs and biological response modifiers.

Radiation therapy has a major role in the treatment of most tumor typesand can increase the cure rate or prolong disease-free survival.Radiation therapy may also be useful in the treatment of recurrences inpatients treated initially with surgery alone. Chemotherapy may be usedbefore, during, or after surgery and radiation therapy. Recurrent tumorsare treated with chemotherapy as well. Anti-proliferative agents used inthe treatment of brain cancers include cisplatin. Examples of thetoxicities associated with this anti-proliferative agent are discussedelsewhere herein.

Restenosis

Restenosis is a form of chronic vascular injury leading to vessel wallthickening and loss of blood flow to the tissue supplied by the bloodvessel. This inflammatory disease can occur in response to vascularreconstructive procedures including any manipulation that relievesvessel obstruction. Thus, restenosis has been a major restrictive factorlimiting the effectiveness of these procedures.

The present discussion comprises prevention or treatment of restenosis,for example by administering to a blood vessel a therapeuticallyeffective amount of the combination of an oligonucleotide therapeuticand an anti-inflammatory agent. Suitable compositions include apolymeric carrier that can be surgically implanted at a restenosis site,or potential restenosis site, or can be injected via a catheter as apolymeric paste or gel. Suitable compositions may comprisepurified/modified fucans discussed herein.

Arthritis

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory diseasecharacterized by pain, swelling, synovial cell proliferation (pannusformation) and destruction of joint tissue. In the advanced stage, thedisease often damages critical organs and may be fatal. The diseaseinvolves multiple members of the immune system (macrophages/monocytes,neutrophils, B cells and T cells) complex cytokine interactions andsynovial cell malfunction and proliferation. Early aggressive treatmenthas been recommended with disease modifying anti-rheumatic drugs(DMARDs) such as methotrexate, which drug is discussed elsewhere herein.

Crystal induced arthritis has been characterized by crystal inducedactivation of macrophages and neutrophils in the joints and is followedby excruciating pain for many days. The disease progresses so that theintervals between episodes gets shorter and morbidity for the patientincreases. This disease has been generally treated symptomatically withnon-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac sodium(Voltaren®). This anti-inflammatory agent has toxicities which includecentral nervous system toxicities such as dizziness and headache;dermatologic toxicities such as rash and pruritus; gastrointestinaltoxicities such as exacerbated ulcerative colitis and Crohn's disease;genitourinary toxicities such as acute renal failure and renal papillarynecrosis; hematologic toxicities such as agranulocytosis, leukopenia andthrombocytopenia; hepatic toxicities such as elevated livertransaminases and hepatitis; and, other toxicities such as asthma andanaphylaxis.

The present discussion comprises prevention or treatment of rheumatoidarthritis, for example via administering to a patient a therapeuticallyeffective amount of an oligonucleotide therapeutic and optionally ananti-inflammatory agent. Suitable compositions include a polymericcarrier that can be injected into a joint as a controlled releasecarrier of the anti-inflammatory agent and microparticulates ascontrolled release carriers of the oligonucleotide therapeutic (which inturn has been incorporated in the polymeric carrier). Suitablecompositions may comprise purified/modified fucans discussed herein.Such polymeric carriers may take the form of polymeric microspheres,pastes or gels.

Inflammatory Conditions

The compositions, etc., herein may optionally inhibit or treatinflammatory conditions involving neutrophils for example comprisingadministering to a patient compositions containing an oligonucleotidetherapeutic and an anti-inflammatory agent. Examples of such conditionsinclude crystal-induced arthritis; osteoarthritis; non-rheumatoidinflammatory arthritis; mixed connective tissue disease; Sjögren'ssyndrome; ankylosing spondylitis; Behçet's syndrome; sarcoidosis;psoriasis; eczema; inflammatory bowel disease; chronic inflammatory lungdisease; neurological disorders; and, multiple sclerosis. Some of thesediseases are discussed further in the following paragraphs.

Chronic Inflammatory Skin Diseases (Including Psoriasis and Eczema)

Psoriasis is a common, chronic inflammatory skin disease characterizedby raised, thickened and scaly lesions which itch, burn, sting and bleedeasily. While these diseases have cellular proliferation and angiogeniccomponents in later stages of the disease, patients often haveaccompanying arthritic conditions. Symptoms may be treated withsteroidal anti-inflammatory agents such as prednisone oranti-proliferative agents such as methotrexate, which agents arediscussed elsewhere herein. The compositions herein may also be used toinhibit or otherwise treat and/or prevent chronic inflammatory skindiseases, for example psoriasis and/or eczema.

The following provides some additional representative examples ofinflammatory diseases that can be treated with compositions discussedherein, include, for example, arterial embolization in arteriovenousmalformations (vascular malformations); menorrhagia; acute bleeding;central nervous system disorders; and, hypersplenism; inflammatory skindiseases such as psoriasis; eczematous disease (atopic dermatitis,contact dermatitis, eczema); immunobullous disease; and, inflammatoryarthritis which includes a variety of conditions including rheumatoidarthritis, mixed connective tissue disease, Sjögren's syndrome,ankylosing spondylitis, Behçet's syndrome, sarcoidosis, crystal inducedarthritis and osteoarthritis (all of which feature inflamed, painfuljoints as a prominent symptom).

Ischemia

Ischemia or ischaemia involves a restriction in blood supply, which mayinclude a shortage of supply of oxygen, glucose and other componentsrequired for proper tissue function, resulting in damage and/ordysfunction of tissue. Ischemia can cause severe problems. For example,tissues can become anoxic, necrotic, and clots can form. Variousattempts have been made to prevent and/or treat ischemia. For example,restoration of blood flow, or reperfusion. Restoration of blood,however, involves the reintroduction of oxygen, which can causeadditional damage due to the production of free radicals, resulting inreperfusion injury. Reperfusion injury can cause severe problems. Thecompositions herein may be used to inhibit or otherwise treat and/orprevent, ischemia, and/or reperfusion injury.

Endotoxemia

Endotoxemia is the presence of endotoxins in the blood. Endotoxemia cancause severe problems. For example, endotoxemia can lead to septicshock. The compositions herein may be used to inhibit, or otherwisetreat and/or prevent, endotoxemia.

Keloid Scarring

Keloid trait causes wounds to heal with raised scars. Keloid traits'raised scars involve abnormal fibrous scarring. Keloid trait causessevere problems, for example, pain and disfigurement. The compositionsherein may be used to inhibit, or otherwise treat and/or prevent, keloidtrait and its resulting raised scars.

Keloid (keloid scar) is a type of scar that expands in growths overnormal skin. Keloids involve abnormal collagen growth, including type Iand type III collage abnormal growth. Keloids cause severe problems, forexample, pain, itchiness, and if infected may ulcerate. Attempts havebeen made to treat or prevent keloids including the use of surgery,dressings, steroid injections and laser therapy. The compositions hereinmay be used to inhibit, or otherwise treat and/or prevent, keloids.

Dermatitis

Dermatitis includes inflammation of the skin including atopic dermatitisand contact dermatitis. For example, contact dermatitis involveslocalized rash and/or irritation of the skin following contact of theskin with a foreign substance. For example, atopic dermatitis is achronically relapsing, pruritic skin disease. Atopic dermatitis issometimes called prurigo Besnier, neurodermitis, endogenous eczema,flexural eczema, infantile eczema, childhood eczema and prurigodiathsique. Eczema is a disease in a form of dermatitis. Other types ofdermatitis include spongiotic dermatitis, seborrhoeic dermatitis(dandruff), dyshidrotic dermatitis (pompholyx), urticaria, vesiculardermatitis (bullous dermatitis), and popular urticaria. Dermatitis cancause severe problems. For example, dry skin, skin rashes, skin edema,skin redness, skin itchiness, skin crusting, cracking, blistering,oozing and bleeding. Attempts have been made to treat or preventdermatitis including the use of corticosteroids and coal tars. Thecompositions herein may be used to inhibit, or otherwise treat and/orprevent, dermatitis including atopic dermatitis, eczema, contactdermatitis, spongiotic dermatitis, seborrhoeic dermatitis, dyshidroticdermatitis, urticaria, vesicular dermatitis, and popular urticaria.

Rosacea

Rosacea is a chronic disease or condition typically characterized byfacial erythema. Rosacea can cause severe problems. For example, rosaceatypically begins as redness on the forehead, nose or cheeks and can alsocause redness on the neck, ears, scalp and chest. For example, rosaceacan cause additional symptoms including telangiectasia, papules,pustules, painful sensations, and in advanced cases rhinophyma (redlobulated nose) may develop. Rosacea subtypes includeerythematotelangiectatic rosacea, papulopustular rosacea, phymatousrosacea, and ocular rosacea. Attempts have been made to treat or preventrosacea including the use of anti-inflammatories and antibiotics. Thecompositions herein may be used to inhibit, or otherwise treat and/orprevent, rosacea including its erythematotelangiectatic, papulopustular,rosacea and ocular subtypes.

Medical Device, Medical Materials, Combination, and PharmaceuticalProducts

The discussion herein also provides medical devices, combination, andpharmaceutical products, comprising compositions as discussed herein ina medical device, combination product or pharmaceutically acceptablecontainer. The products can also include a notice associated with thecontainer, typically in a form prescribed by a governing agencyregulating the manufacture, use, or sale of medical devices,combination, and pharmaceuticals or biopharmaceuticals, whereby thenotice is reflective of approval by the agency of the compositions, suchas a notice that a purified/modified fucan has been approved as ananti-proliferative agent or anti-inflammatory agent, e.g., for human orveterinary administration to treat proliferative diseases orinflammatory diseases (such as, for example, inflammatory arthritis,restenosis, surgical adhesions, psoriasis and peritonitis). Instructionsfor the use of the purified/modified fucan herein may also be included.Such instructions may include information relating to the dosing of apatient and the mode of administration.

The present application is further directed to methods of making thevarious elements of the purified/modified fucan, systems etc., discussedherein, including making the compositions themselves, as well as tomethods of using the same, including for example treatment of theconditions, diseases, etc., herein.

The present application further comprises medical devices, medicalmaterials, medical combination products, and pharmaceutical products fortreatment of fibrous adhesions, arthritis, psoriasis or other diseasesas desired comprising the purified/modified fucan and fucan compositionspresented herein. The materials, etc., can be used in a medicament fortreating fibrous adhesions, such as a surgical adhesions, arthritis,psoriasis or other diseases as desired. Also provided are methods ofmanufacturing and using such medicaments able to reduce symptomsassociated with at least one of fibrous adhesions, arthritis, andpsoriasis in a patient including a human patient, comprising combining apharmaceutically effective amount of a fucan such as fucoidan asdiscussed herein with a pharmaceutically acceptable excipient or buffer.

The following Examples provide exemplary discussions of certainembodiments herein but the disclosure and claims are not limitedthereto.

Example 1: Chemical Structural Modification

An exudate-extract was obtained from Laminaria hyperborea. Theexudate-extract was filtered and small molecules were removed bytangential flow filtration (TFF) over a 100 kDa filter. A sample of theresulting retentate was lyophilized to obtain otherwise unmodifiedsample A. The resulting retentate was brought to 0.25 M NaOH by additionof 10 M NaOH solution and left at room temperature for 16 hours. Theresulting sample was then centrifugally filtered over a 50 kDa filterand the resulting retentate collected and lyophilized to obtainbase-treated sample B. Both unmodified sample A and base-treated sampleB were analyzed by proton nuclear magnetic resonance spectroscopy(¹H-NMR) and the resulting ¹H-NMR spectrum are shown in FIG. 2A.

FIG. 2A demonstrates the chemical structural modification of the fucan:the broad peak with a chemical shift about 2.0 ppm that is present inthe unmodified sample A is not present in the base-treated sample B.

Unmodified sample A and base-treated/modified sample B were furtheranalyzed by 2D ¹H-¹³C heteronuclear multiple quantum coherence (HMQC).The HMQC spectra, shown in FIG. 2B, were acquired at 70° C. with solventsignal suppression on a 600 MHz spectrometer equipped with 5-mm coldprobe. A high number of scans of the HMQC spectra were acquired in therange from 10-30 ppm in the carbon dimension in 8 increments of 256-512scans each; such scans were combined to create the spectra in FIG. 2B.

The HMQC spectra for unmodified sample A has a cross-peak correspondingto 0-acetyl groups, indicated by the signal circled in FIG. 2B. Thiscross-peak is not present in the spectra for base-treated sample B. Thisdemonstrates the removal of acetyl groups from the fucan, and thuschemical structural modification of the fucan in base-treated sample Bby the NaOH treatment.

Example 2: Physically Induced Flocculation

A brown powder-feedstock fucoidan was dissolved at about 10% w/v indistilled water to obtain a starting solution. Sodium chloride was addedto the starting solution, to produce a mixture with a final sodiumchloride concentration of about 0.1 M. The mixture was heated to nearboiling for between 10-15 minutes. Treatment of the mixture at thistemperature induced flocculation of suspended impurities and particulatenon-fucoidan matter. The mixture was centrifuged at 2300 gravities for40 minutes to separate the fucoidan containing solution from theflocculated non-fucoidan components. The fucoidan containing solutionwas visually inspected and a visual decrease in particulate matter andcolor was observed. A lyophilized portion of the fucoidan containingsolution comprised an off-white powder with significantly less colorthan the feedstock fucoidan used. The loss of color can be quantifiedand compared, for example, by obtaining a ultraviolet/visible (UV/Visspectrum) of the feedstock fucoidan at 10 mg/mL in water and a UV/Visspectrum of the purified/modified fucan at 10 mg/mL in water,determining the total absorbance in the visible region of the spectrum,which is between about 400 nm and about 700 nm, and observing a decreaseof about at least 5%, 10% or 20% in total absorbance in thepurified/modified fucan relative to the feedstock fucoidan.

Example 3: Solid Phase Extraction

A brown powder-feedstock fucoidan was added to a 40 degrees Celsiusmixture of 0.5 M NaOH in 70% v/v ethanol/water. The resulting reactionmixture was stirred and maintained at 40 degrees Celsius for 2 hours.The reaction mixture was then centrifuged to separate the solidpurified/modified fucoidan from the 0.5 M NaOH in 70% v/v ethanol/watersupernatant containing the extracted impurities.

The solid purified/modified fucoidan was found to be contain noticeablyless color to the human eye than the feedstock fucoidan when visualized.This loss of color indicated the removal of impurities, such asphlorotannins, because fucans do not contain a chromophore and so willbe colorless when completely pure. The loss of color can be quantifiedand compared, for example, by obtaining a ultraviolet/visible (UV/Visspectrum) of the feedstock fucoidan at 10 mg/mL in water and a UV/Visspectrum of the purified/modified fucan at 10 mg/mL in water,determining the total absorbance in the visible region of the spectrum,which is between about 400 nm and about 700 nm, and observing a decreaseof about at least 5%, 10% or 20% in total absorbance in thepurified/modified fucan relative to the feedstock fucoidan.

Example 4: Chemically Induced Precipitation

A feedstock fucoidan composition was dissolved at 15% w/v in distilledwater to form a starting solution. The starting solution was found tocontain suspended particulates by observation. Calcium chloride wasadded to the starting solution to a level of 0.5 M to produce a reactionmixture. To simulate known impurities in natural fucoidan, sodiumalginate was added at a concentration of 5% w/w alginate/fucoidan andstarch was added at a concentration of 5% w/w starch/fucoidan. Starchwas used as a mimetic for laminarin in this case. 10 M NaOH was addeddropwise to the reaction mixture to bring the pH to between 7 and 8.This was done to avoid degradation of the fucoidan in the reactionmixture. A minimal amount of 10 M NaOH was again added to the reactionmixture to avoid the acidification of the reaction mixture from thesubsequent addition of phosphoric acid. The reaction mixture was broughtto 0.5 M phosphate through the addition of phosphoric acid. Thisinitiated flocculation of the suspended particulates and precipitatedimpurities via the action of the calcium phosphate formed by thereaction of the calcium chloride with the phosphoric acid. The reactionmixture was allowed to stand at room temperature for 10 minutes to allowthe flocculation to continue. The reaction mixture was centrifuged at17568 gravities for 17 minutes to separate the desired purified fucoidanin a supernatant solution from the flocculated impurities. Thesupernatant solution was visually inspected to qualitatively assess theremoval of color and particulate. An aliquot of the supernatant solutionwas also analyzed by UV/Vis absorption in the 300-800 nm region toassess the removal of non-fucoidan components that scatter light and/orabsorb light in the UV/Vis spectral region. An aliquot of thesupernatant solution was also lyophilized to obtain the fucan content.An aliquot of the supernatant solution was also hydrolyzed in 3M HCl at90 degrees Celsius and analyzed by High Performance AnionExchange—Pulsed Amperometry Detection (HPAE-PAD) for the detection oftotal carbohydrates and assessment of the removal of laminarin andalginate. Quantification of the impurities was against standards ofmonomeric glucose to assess removal of laminarin and monomericmannuronic acid and monomeric guluronic acid to assess the removal ofalginates.

Analysis results from the starting fucoidan and the resultingpurified/modified fucans are presented in table 1 below.

TABLE 1 Analytical results of starting fucoidan and treated solutionsFlocculation Alginate by Starch by UV/Vis signal Treatment Visualappearance HPAE-PAD HPAE-PAD 300-800 nm Starting Brown solid, opaqueNone detected None detected 670.587* fucoidan brown in solution TreatedLight brownish yellow None detected None detected 9.616 fucoidan clearsolution *Values determined via a representative starting fucoidan

Example 5: Chemically Induced Precipitation

A feedstock fucoidan composition was dissolved at 15% w/v in distilledwater to form a starting solution. The starting solution was found tocontain suspended particulates by observation. To simulate knownimpurities in natural fucoidan, sodium alginate was added at aconcentration of 5% w/w alginate/fucoidan and starch was added at aconcentration of 5% w/w starch/fucoidan. Starch was used as a mimeticfor laminarin in this case. 10 M NaOH was added dropwise to the startingsolution to bring the pH to between 7 and 8. This was done to avoiddegradation of the fucoidan in the solution in case the subsequentaddition of aluminum sulfate were to render the starting solutionacidic. The starting solution was brought to 0.1 M aluminum sulfate toproduce a reaction mixture. This initiated precipitation of impuritiesas well as flocculation of impurities and suspended particulates by thesimultaneously formed aluminum hydroxide. The reaction mixture wasallowed to stand at room temperature for 10 minutes to allow theflocculation to continue. The reaction mixture was centrifuged at 17568gravities for 17 minutes to separate the desired purified fucoidan in asupernatant solution from the flocculated impurities. The supernatantsolution was visually inspected to qualitatively assess the removal ofcolor and particulate. An aliquot of the supernatant solution was alsoanalyzed by UV/Vis absorption in the 300-800 nm region to assess theremoval of non-fucoidan components that scatter light and/or absorblight in the UV/Vis spectral region. An aliquot of the supernatantsolution was also lyophilized to obtain the fucan content. An aliquot ofthe supernatant solution was also hydrolyzed in 3M HCl at 90 degreesCelsius and analyzed by High Performance Anion Exchange—PulsedAmperometry Detection (HPAE-PAD) for the detection of totalcarbohydrates and assessment of the removal of laminarin and alginate.Quantification of the impurities was against standards of monomericglucose to assess removal of laminarin and monomeric mannuronic acid andmonomeric guluronic acid to assess the removal of alginates.

Analysis results from the starting fucoidan and the resultingpurified/modified fucans are presented in table 2 below.

TABLE 2 Analytical results of starting fucoidan and treated solutionsFlocculation Alginate by Starch by UV/Vis signal Treatment Visualappearance HPAE-PAD HPAE-PAD 300-800 nm Starting Brown solid, opaqueNone detected None detected 670.587* fucoidan brown in solution TreatedLight yellow clear None detected None detected 15.188 fucoidan solution*Values determined via a representative starting fucoidan

Example 6: Chemically Induced Precipitation

A starting fucoidan composition was dissolved at 15% w/v in distilledwater to form a starting solution. The starting solution was found tocontain suspended particulates by observation. To simulate knownimpurities in natural fucoidan, sodium alginate was added at aconcentration of 5% w/w alginate/fucoidan and starch was added at aconcentration of 5% w/w starch/fucoidan. Starch was used as a mimeticfor laminarin in this case. Calcium chloride was added to the startingsolution to a level of 0.5 M to produce a reaction mixture. Thisinitiated the precipitation of the alginate. 10 M NaOH was addeddropwise to the reaction mixture to bring the pH to between 7 and 8.This was done to avoid degradation of the fucoidan in the reactionmixture. The reaction mixture was brought to 0.5 M aluminum sulfate.This initiated flocculation of the suspended particulates, calciumalginate precipitate and other impurities via the action of calciumsulfate formed by the reaction of calcium chloride with aluminum sulfateand by the action of aluminum hydroxide formed from the aluminum sulfatein the reaction mixture. The reaction mixture was allowed to stand atroom temperature for 10 minutes to allow the flocculation to continue.The reaction mixture was centrifuged at 17568 g for 17 minutes toseparate the desired purified fucoidan in a supernatant solution fromthe flocculated impurities. The supernatant solution was visuallyinspected to qualitatively assess the removal of color and particulate.An aliquot of the supernatant solution was also analyzed by UV/Visabsorption in the 300-800 nm region to assess the removal ofnon-fucoidan components that scatter light and/or absorb light in theUV/Vis spectral region. An aliquot of the supernatant solution was alsolyophilized to obtain the fucan content. An aliquot of the supernatantsolution was also hydrolyzed in 3M HCl at 90 degrees Celsius andanalyzed by High Performance Anion Exchange—Pulsed Amperometry Detection(HPAE-PAD) for the detection of total carbohydrates and assessment ofthe removal of laminarin and alginate. Quantification of the impuritieswas against standards of monomeric glucose to assess removal oflaminarin and monomeric mannuronic acid and monomeric guluronic acid toassess the removal of alginates.

Analysis results from the starting fucoidan and the resultingpurified/modified fucans are presented in table 3 below.

TABLE 3 Analytical results of starting fucoidan and treated solutionsFlocculation Alginate by Starch by UV/Vis signal Treatment Visualappearance HPAE-PAD HPAE-PAD 300-800 nm Starting Brown solid, opaqueNone None 670.587* fucoidan brown in solution detected detected TreatedLight yellow clear solution None None 23.814 fucoidan detected detected*Values determined via a representative starting fucoidan

Example 7: Liquid-Liquid Extraction

A starting fucan composition containing impurities is dissolved at 10mg/mL in distilled water to produce an aqueous starting solution. 20%v/v heptane is added to the aqueous starting solution containing thestarting fucoidan composition and the organic-aqueous mixture is thenmixed at high shear for 30 minutes. The mixing is terminated, and theorganic-aqueous mixture placed in a separatory funnel for the separationof the organic phase from the aqueous phase. The denser aqueous phasecontaining the desired fucan component settles to the bottom of theseparatory funnel while the less dense organic phase containingimpurities is present at the upper end of the separatory funnel. Theorganic-aqueous mixture is allowed to sit in the separatory funnel for10 minutes. The aqueous phase is then decanted and collected as thedesired purified/modified fucan in solution. The purified/modified fucanin solution can be found to contain between about 30%, 50%, 70% to about100% less lipids, fatty acids, phlorotannin, proteins, fucoxanthinand/or chlorophyll than the starting fucan composition.

Example 8: Liquid-Liquid Extraction

A starting fucan composition containing impurities is dissolved at 10mg/mL in distilled water to produce an aqueous starting solution. 20%v/v 1-butanol is added to the aqueous starting solution containing thestarting fucoidan composition and the organic-aqueous mixture is thenmixed at high shear for 30 minutes. The mixing is terminated, and theorganic-aqueous mixture placed in a separatory funnel for the separationof the organic phase from the aqueous phase. The denser aqueous phasecontaining the desired fucan component settles to the bottom of theseparatory funnel while the less dense organic phase containingimpurities is present at the upper end of the separatory funnel. Theorganic-aqueous mixture is allowed to sit in the separatory funnel for10 minutes. The aqueous phase is then decanted and collected as thedesired purified/modified fucan in solution. The purified/modified fucanin solution can be found to contain between about 30%, 50%, 70% to about100% less lipids, fatty acids, phlorotannin, proteins, fucoxanthinand/or chlorophyll than the starting fucan composition.

Example 9: Liquid-Liquid Extraction

A starting fucan composition containing impurities is dissolved at 10mg/mL in distilled water to produce an aqueous starting solution. 20%v/v ethyl acetate is added to the aqueous starting solution containingthe starting fucoidan composition and the organic-aqueous mixture isthen mixed at high shear for 30 minutes. The mixing is terminated, andthe organic-aqueous mixture placed in a separatory funnel for theseparation of the organic phase from the aqueous phase. The denseraqueous phase containing the desired fucan component settles to thebottom of the separatory funnel while the less dense organic phasecontaining impurities is present at the upper end of the separatoryfunnel. The organic-aqueous mixture is allowed to sit in the separatoryfunnel for 10 minutes. The aqueous phase is then decanted and collectedas the desired purified/modified fucan in solution. Thepurified/modified fucan in solution can be found to contain betweenabout 30%, 50%, 70% to about 100% less lipids, fatty acids,phlorotannin, proteins, fucoxanthin and/or chlorophyll than the startingfucan composition.

Example 10: Diafiltration

A starting solution comprising about 8% w/v or a starting fucoidancomposition was provided. The starting solution was filtered through a0.22 micron filter. The cationic content in an aliquot of the filteredstarting solution were determined by inductively coupled plasma massspectrometry (ICP-MS) and found to contain above 0.01% w/waluminum/fucan, above 10⁻⁵% w/w arsenic/fucan and above 0.01% w/wcalcium/fucan, all being undesirable levels of each respective cation.The starting solution was diafiltered for 4 diavolumes with 0.1 M EDTA,0.01 M NaOH solution. The resulting retentate fucoidan solution was thendiafiltered with about 2.5 diavolumes of 5 mM Na₂SO₃, 5 mM NaClsolution. The resulting secondary retentate fucoidan solution wasassayed for cationic content by ICP-MS. The results for the startingfucoidan composition and the resulting purified/modified fucan are shownin Table 4 below.

TABLE 4 Analytical results of starting fucoidan and treated solutions %w/w % w/w % w/w % w/w Treatment Al/fucoidan As/fucoidan Ca/fucoidanMg/fucoidan Input 2.95 × 10⁻¹ 1.79 × 10⁻⁵ 2.78 × 10⁻² 4.76 × 10⁻³Purified/ 3.33 × 10⁻⁴ <4.38 × 10⁻⁶ 3.85 × 10⁻³ 1.70 × 10⁻³ modifiedFucan

Example 11: Super Critical Fluid Extraction

A solid starting fucoidan composition of about 100 g is provided. Thesolid is placed in a supercritical extractor. The extractor ispressurized to 5800 psi and heated to 50 degrees Celsius and then purgedwith supercritical carbon dioxide at 100 mL/minute for 3 hours. Thesupercritical carbon dioxide is purged from the extractor and the solidmodified/purified fucoidan collected and analyzed for impurities. Thecollected solid modified/purified fucoidan can be found to containbetween about 30% to about 100% less lipids, fatty acids, phlorotannins,laminarins, alginates, proteins, Maillard reaction products,fucoxanthin, chlorophyll, free ions, bacteria and/or DNA than thestarting fucoidan composition.

Example 12: Chemically Induced Precipitation, Lysis and Flocculation

A starting fucoidan composition, found to contain about 0.70% w/w totalnitrogen, was dissolved at 15% w/v in distilled water to form a startingsolution. The presence of total nitrogen indicates the presence ofundesired impurities, for example, cellular components, DNA, proteinsand bacteria. A reduction in the total nitrogen indicates that thesenitrogen-containing impurities have been removed from the startingfucoidan composition or from the fucoidan polymers, as the case may be,because the nitrogen containing impurities may be chemically orionically bound to such fucoidan molecules.

The starting solution was found to contain suspended particulates byobservation. Calcium chloride was added to the starting solution to alevel of 0.5 M to produce a reaction mixture. This initiated theprecipitation of a solid portion suspected to contain impurities. About15 mL of 10 M NaOH was added dropwise to the reaction mixture to bringthe pH to between 7 and 8. This was done to avoid degradation of thefucoidan in the reaction mixture. The reaction mixture was brought to0.5 M phosphate through the addition of phosphoric acid. This initiatedflocculation of the suspended particulates and precipitated impuritiesvia the action of calcium phosphate formed by the reaction of thecalcium chloride with the phosphoric acid. The reaction mixture wascentrifuged at 33,746 gravities for 5 minutes to separate a firstpurified/modified fucoidan in a supernatant solution from theflocculated impurities. The first purified/modified fucoidan was foundto contain about 0.10% w/w total nitrogen. A portion of the firstpurified/modified fucoidan in the supernatant solution was furtherpurified by diafiltration over a 100 kDa MWCO centrifugal filter against6 diavolumes of 5 mM NaCl. The resulting first retentatepurified/modified fucoidan was found to contain about 0.08% w/w totalnitrogen.

A second portion of the first purified/modified fucoidan was furtherprocessed by adding 1 M sodium dodecyl sulfate solution as cellulardisrupting agent to a concentration of 0.010 M. 10 M NaOH solution wasadded to a concentration of 0.26 M to render the mixture basic. Theresulting reaction mixture was stirred for about 30 minutes at roomtemperature to obtain a cloudy light brown mixture.

After about 30 minutes, 45% w/v KOH solution was added to aconcentration of about 0.04 M. The addition of potassium resulted in theprecipitation of SDS and undesired impurities along with the SDS. 48%w/v aluminum sulfate solution was added to a concentration of about 0.06M. The formation of aluminum hydroxide flocculated undesired impuritiesin the reaction mixture. Sodium sulfite solid was added and dissolved toa concentration of 0.02 M to quench potential oxidants in the reactionmixture.

The resulting reaction mixture was stored in a refrigerator for about 16hours, followed by centrifugation at 33,746 gravities for 5 minutes toseparate a second purified/modified fucoidan in a supernatant solutionfrom the flocculated impurities. The second purified/modified fucoidanwas found to contain about 0.06% w/w total nitrogen. A portion of thesecond purified/modified fucoidan was further processed by diafiltrationover a 100 kDa MWCO centrifugal filter against 6 diavolumes of 5 mM NaClto provide a second retentate purified/modified fucoidan. The resultingsecond retentate purified/modified fucoidan was found to contain about0.03% w/w total nitrogen.

Example 13: Preparation of Five Purified/Modified Fucans

The methods discussed herein can be used, combined, modified andpermuted in any manner to obtain purified/modified fucans. Fivepurified/modified fucans were prepared using a combination of chemicallyinduced precipitation and diafiltration discussed in examples 4, 5, 6and 10 to evaluate the efficacy of purified/modified fucans in medicaland surgical applications. These 5 fucans are referred to as fucan 1 tofucan 5 herein. Fucan 1 and fucan 2 were produced at about a 2 kg scaleusing the methods discussed in example 4 and example 10. Fucan 3 andfucan 5 were produced at about a 30 g scale using the methods discussedin example 4 and example 10. Fucan 4 was produced at about a 1 kg scaleusing the methods discussed in example 5 and example 10. Fucans 1 to 5were converted into solid purified/modified fucans by diafiltrationagainst a low conductivity salt solution followed by lyophilization toobtain white solids. Two additional fucans were extracted from brownseaweed, herein referred to as fucan 6 and fucan 7. Fucan 6 was providedas a solid composition by FMC BioPolymer®. None of the processesdiscussed above were used in the production of fucan 6. Fucan 7 wasextracted from brown seaweed by near boiling HCl. Some impurities wereremoved by selective precipitation using ethanol as the precipitant.After the selective precipitation, the fucan was collected as a solidcomposition by further precipitation of the fucan with ethanol,centrifugation and lyophilization. Fucan 7 was further treated with themethod discussed in example 3, then dissolved in water and diafilteredagainst deionized water before being lyophilized to obtain fucan 7 as asolid composition. The fucose, galactose, sulfate and total counterionlevels of fucan 1 to fucan 7 are determined as discussed in examples 14and example 15 below. These fucans 1-7 are discussed further below, forexample in example 16 and Table 6.

Example 14: Measurement of Corrected Fucose Content and CorrectedGalactose Content of Fucan 1 to Fucan 7

Solid fucan compositions were dissolved in 72% w/w sulfuric acid at 40mg/mL and incubated at 45° C. in a water bath for 30 minutes. The acidhydrolysate was then diluted to 4% w/w sulfuric acid in a high-pressuretube and incubated at 120° C. for 60 minutes. The resulting second acidhydrolysate was diluted to a 1/333 concentration with distilled waterand run on high performance anionic exchange column chromatography setup with pulsed amperometry detection (HPAE-PAD). Separation of analyteswas accomplished by running 10 mM NaOH eluent at 1.0 mL/minute using anisocratic pump.

The uncorrected fucose content of the fucans were determined byinterpolation on a standard curve for fucose. The uncorrected galactosecontent of the fucans were determined by the method of standardaddition. Corrected fucose content was determined by accounting for theaddition of one molecule of water upon hydrolysis of a glycosidic bond,and accounting for the addition of two hydroxyl group upon hydrolysis oftwo sulfate-ester bonds pet fucose. Corrected galactose content wasdetermined by accounting for the addition of one molecule of water uponhydrolysis of a glycosidic bond.

The results from this analysis are shown in table 5 below.

Example 15: Measurement of Total Sulfate Content, Total CounterionContent and Total Water Content of Fucan 1 to Fucan 7

Solid fucan compositions were dissolved in deionized water, hydrolyzedunder acidic conditions and analyzed by ICP-MS for % w/w total sulfurand counterions content. Sulfur content was converted to sulfate contentby multiplying the sulfur content by the molar ratio of sulfate tosulfur to obtain % w/w sulfate content of the purified/modified fucan.The counterions observed in the purified/modified fucan discussed hereinincluded potassium and sodium counterions. The results from thisanalysis are shown in table 5 below.

The % w/w results for total corrected fucose, corrected galactose andsulfate are also shown in Table 5 below. The results for total fucose,galactose and sulfate are determined by adding the corrected fucose,corrected galactose and sulfate values together, a more detailed andcomplete calculation including aspects discussed above shown in equation1 below. The total counterion content, is determined by adding togetherthe total sodium and total potassium content.

$\begin{matrix}{{{Total}{fucose}},{{{galactose}{and}{sulfate}\left( {\%{w/w}{of}{fucan}} \right)} = {{{Total}{fucose}{hydrolysate}\left( {\%{w/w}} \right)*\frac{{16{4.1}6} - {5{2.0}0}}{16{4.1}6}} + {{Total}{galactose}{hydrolysate}\left( {\%{w/w}} \right)*\frac{{18{0.1}6} - {1{8.0}0}}{18{0.1}6}} + {{Total}{sulfur}\left( {\%{w/w}} \right)*\frac{9{6.0}6}{3{2.0}6}}}}} & {{Equation}1}\end{matrix}$

TABLE 5 Components as % w/w of the fucan Total Total fucose, fucose,galactose, galactose Total sulfate and Fucose Galactose Sulfate andcounterion counterion % % % sulfate Na % K % content % content w/w w/ww/w % w/w w/w w/w w/w % w/w Fucan 1 34.3 2.9 44.9 82.2 11.4 0.7 11.694.3 Fucan 2 34.7 2.5 41.7 78.9 9.8 0.8 10.6 89.5 Fucan 3 35.3 2.3 41.779.2 9.4 1.6 10.9 90.2 Fucan 4 31.1 3.4 51.3 84.0 7.8 5.3 13.0 98.8Fucan 5 32.7 3.0 40.6 76.3 11.0 1.3 12.3 88.6 Fucan 6 18.6 1.4 11.4 31.38.3 1.4 9.7 41.0 Fucan 7 23.3 5.9 11.4 40.6 2.6 0.9 3.5 44.1

Table 5 demonstrates that purified/modified fucans with less than about12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% w/w impurities can beprepared using methods discussed herein.

Table 5 further demonstrates purified/modified fucans with a totalgalactose, fucose and sulfate content of between about 77% w/w and about87% w/w have been produced.

Table 5 further demonstrates purified/modified fucans with a totalcounterion content between about 9% w/w and about 14% w/w of the fucan.

The total water content of fucan 1, fucan 3, fucan 4 and fucan 5 weredetermined by loss on drying (LOD) at 104° C. The total water contentwere determined to be 3.8%, 2.4%, 3.2% and 4.7% w/w of the respectivepurified/modified fucans.

Example 16: Measurement of Molecular Weight Distributions of Fucan 3 andFucan 4

Gel permeation chromatography (GPC) was used to evaluate the molecularweight distributions obtained for the purified/modified fucans fucan 3and fucan 4. There are a large number of different parameters, columnsand standards available for use in gel permeation chromatography,resulting in a variety of instrumentation set-ups available for theanalysis of molecular weight. For molecular weight determinationsherein, the GPC were conducted using the following parameters: Themobile phase was 0.1M sodium nitrate run at 0.6 mL/min. The columncompartment and detector were at 30° C. A Waters 2414 refractive indexdetector was used for detection.

Suitable GPC columns include GPC columns compatible with aqueoussolvents, for example columns packed with at least one of sulfonatedstyrene-divinylbenzene, NH-functionalized acrylate copolymer network,modified silica and hydroxylated polymethacrylate-based gel. For theanalyses herein, three columns were used in series, comprising one 40 mmlong guard column with an inner diameter (ID) of 6 mm packed with 6 μmparticle size hydroxylated polymethacrylate-based gel, followed by afirst 300 mm analytical GPC column with a 7.8 mm ID packed with 12 μmparticle size hydroxylated polymethacrylate-based gel that has anexclusion limit of about 7,000 kDa and an effective molecular weightrange of between about 50 kDa and about 5,000 kDa, followed by a second300 mm analytical GPC column with a 7.8 mm ID packed with 10 μm particlesize hydroxylated polymethacrylate-based gel that has an exclusion limitof about 7,000 kDa and an effective molecular weight range of betweenabout 1 kDa and about 6,000 kDa. The total effective molecular weightrange of the column set up was between about 1 kDa and about 6,000 kDa.An example of this column set up can be Ultrahydrogel®guard-Ultrahydrogel® 2000-Ultrahydrogel® Linear columns connected inseries.

Samples run were quantified against a standard curve comprising oftraceable standards from the American Polymer Standards Corporation:DXT3755K (peak molecular weight=2164 kDa), DXT820K (peak molecularweight=745 kDa), DXT760K (peak molecular weight=621 kDa), DXT670K (peakmolecular weight=401 kDa), DXT530K (peak molecular weight=490 kDa),DXT500K (peak molecular weight=390 kDa), DXT270K (peak molecularweight=196 kDa), DXT225K (peak molecular weight=213 kDa), DXT150K (peakmolecular weight=124 kDa), DXT55K (peak molecular weight=50 kDa), DXT50K(peak molecular weight=44 kDa) and DXT5K (peak molecular weight=4 kDa),the peak molecular weights of these standards being between about 4 kDaand about 2,200 kDa. The standard curve used may, for example, includeDextran 3755 kDa, at least one of Dextran 50 kDa and Dextran 55 kDa, andbetween 3 to 6 additional traceable standards discussed herein, thecalibration points being the peak molecular weights of the calibrantsused. An example calibration curve may consist of DXT3755K, DXT 820K,DXT530K, DXT500K, DXT225K and DXT55K. The columns used herein had atotal effective molecular weight range that encompassed and extendedbeyond the peak molecular weight range of the standards used forquantification of the fucans.

Results in table 6 below contain abbreviations used for certaincharacteristics of a molecular weight distribution. Gel permeationchromatography is denoted by GPC, peak molecular weight is denoted byPMW, weight average molecular weight is denoted by WAMW, number averagemolecular weight is denoted by NAMW, percentage distribution is denotedby % dist. and molecular weight is denoted by MW.

TABLE 6 Molecular weight distribution characteristics of two modifiedfucans PMW WAMW NAMW % dist. % dist. % dist. (kDa) (kDa) (kDa) >100kDa >200 kDa >500 kDa Fucan 3 690.98 1166.60 443.37 97.77 90.06 63.89Fucan 4 686.21 1876.74 524.89 98.37 92.97 69.90

Example 17: Preparation of Highly Purified Fucan Composition by Drying

About 100 mg of Fucan 1 was placed in a crucible. The cruciblecontaining Fucan 1 was placed in an oven at 105° C. for 30 minutes toproduce a further-purified fucan composition, hereinafter called Fucan1′. The crucible containing the further-purified Fucan 1′ compositionwas removed from the oven and placed in a desiccator. Thefurther-purified Fucan 1′ composition is analyzed under a moisture freeatmosphere for total fucose and galactose by HPAE-PAD and for totalsulfur and counterion content by ICP-MS, and is found to contain a totalfucose, galactose, sulfate and counterion content of over 99.9% w/w, inother words, less than 0.1% impurities.

Example 18: Preparation of Highly Purified Fucan Composition by Drying

About 600 mg of Fucan 1 was placed on an aluminum pan in an Ohaus MB 90moisture analyzer instrument. The instrument was programmed to heatFucan 1 at 105° C. for 30 minutes to produce a further-purified Fucan 1″composition. The further-purified Fucan 1″ composition was removed fromthe instrument and placed in a desiccator. The sample is analyzed undera moisture free atmosphere for total fucose and galactose by HPAE-PADand for total sulfur and counterion content by ICP-MS, and is found tocontain a total fucose, galactose, sulfate and counterion content ofover 99.9% w/w, less than 0.1% impurities.

Example 19: Uterine Horn Fibrous Adhesion Treated with Fucan 1

To determine the efficacy of the purified/modified fucan 1 in inhibitingsurgical adhesions, the following double uterine horn (DUH) surgerieswere performed on both horns of a total of two New Zealand Whiterabbits. Prior to surgery, the rabbits were weighed and then preparedfor surgery by premedication with ketamine and xylazine.

Fucoidan solution was prepared at 0.33 mg/mL in Lactated RingersInjection USP (LRS), sterilizing by filtration. All instruments weresterile, and a sterile field was maintained throughout the surgeries.The abdomen was cleaned and entered via a midline abdominal incision.The uterine horns were located, exteriorized and scraped to inducedamage. The abdominal wall near the scraped uterine horns was alsoscraped. A minimal amount of fucoidan solution was applied directly tothe injured uterine horns and sidewall areas. The damaged uterine hornsand abdominal wall were placed next to each other and stabilized withsutures. 15 mL/kg fucoidan solution per rabbit weight was applied to theabdominal cavity before the incision was closed. Adhesion was evaluatedtwo weeks after the surgery. Length of the uterine horn adhesion wasmeasured with a ruler. The uterine horn adhesion coverage percentage,being the length of the adhesion as a percentage of the total damageduterine horn length was calculated as:Adhesion coverage (%)=100×uterine horn adhesion length÷total damageduterine horn length  Equation 2:

The same surgical method was applied to New Zealand White rabbits,receiving 15 mL/kg of Lactated Ringer's Injection USP (LRS) instead offucoidan solution as a control group. The control group receiving LRSwas determined to have a 63% adhesion coverage using equation 2. Table 7shows the results obtained using the method described above for fucan 2,being a representative example of a purified/modified fucan. The resultsin the table below are shown as the reduction in adhesion coveragerelative to the control group.

Table 7 provides the results of treating six uterine horn with fucan 1.

TABLE 7 Reduction in rabbit uterine horn adhesion using fucan 1 DoseNumber of % Reduction in uterine horn (mg/kg) Uterine Horns adhesioncoverage vs. control Fucan 5 6 100% 1

As can be seen from the results of Table 7, the purified/modified fucansdiscussed herein can be used to successfully treat post-surgical uterinehorn adhesions.

Example 20: Uterine Horn Fibrous Adhesion Treated with Fucan 4

To determine the efficacy of the purified/modified fucan 4 in inhibitingsurgical adhesions, the following double uterine horn (DUH) surgerieswere performed on both horns of a total of four New Zealand Whiterabbits. Prior to surgery, the rabbits were weighed and then preparedfor surgery by premedication with ketamine and xylazine.

Fucoidan solution was prepared at 3.75 mg/mL in Lactated RingersInjection USP (LRS), sterilizing by filtration. All instruments weresterile, and a sterile field was maintained throughout the surgeries.The abdomen was cleaned and entered via a midline abdominal incision.The uterine horns were located, exteriorized and scraped to inducedamage. The abdominal wall near the scraped uterine horns were alsoscraped. 4 mL of fucoidan solution was applied directly to the leftinjured uterine horn and sidewall area and 4 mL of fucoidan solution wasapplied directly to the right injured uterine horn and sidewall area.The damaged uterine horns and abdominal wall were placed next to eachother and stabilized with sutures. A drainage tube was positioned in theperitoneal cavity before the incision was closed. The drainage tube isremoved 48 hours post-surgery. Adhesion was evaluated two weeks afterthe surgery. Length of the uterine horn adhesion was measured with aruler. The uterine horn adhesion coverage was calculated using equation2.

The same surgical method was applied to 3 New Zealand White rabbits,receiving 4 mL per side of Lactated Ringer's Injection USP (LRS) insteadof fucoidan solution as a control group. The control group receiving LRSwas determined to have a 73% adhesion coverage using equation 2. Table 8shows the results obtained using the method described above for fucan 4,being a representative example of a purified/modified fucan. The resultsin the table below are shown as the reduction in adhesion coveragerelative to the control group.

Table 8 provides the result of treating eight uterine horns with fucan4.

TABLE 8 Reduction in rabbit uterine horn adhesion using fucan 4 DoseNumber of % Reduction in uterine horn (mg/kg) Uterine Horns adhesioncoverage vs. control Fucan 9.8 8 92.9% (i.e., 92.9% reduction 4 infibrous adhesions compared to control)

As can be seen from the results of Table 8, the purified/modified fucanscan be used to successfully treat post-surgical uterine horn adhesions.

Example 21: Uterine Horn Fibrous Adhesion Treated with Fucan 6

To determine the efficacy of the purified/modified fucan 6 in inhibitingsurgical adhesions, the following double uterine horn (DUH) surgerieswere performed on both horns of a total of four New Zealand Whiterabbits. Prior to surgery, the rabbits were weighed and then preparedfor surgery by premedication with ketamine and xylazine.

Fucoidan solution was prepared at 0.33 mg/mL in Lactated RingersInjection USP (LRS), sterilizing by filtration. All instruments weresterile, and a sterile field was maintained throughout the surgeries.The abdomen was cleaned and entered via a midline abdominal incision.The uterine horns were located, exteriorized and scraped to inducedamage. The abdominal wall near the scraped uterine horns was alsoscraped. The damaged uterine horns and abdominal wall were placed nextto each other and stabilized with sutures. About 15 mL/kg fucoidansolution per rabbit weight was applied to the abdominal cavity beforethe incision was closed. Adhesion was evaluated two weeks after thesurgery. Three rabbits were evaluated at each fucoidan concentrationprepared. Length of the uterine horn adhesion was measured with a ruler.The uterine horn adhesion length was calculated using equation 2.

The same surgical method was applied to 4 New Zealand White rabbits,receiving about 15 mL/kg of control Lactated Ringer's Injection USP(LRS) instead of fucoidan solution. The control group receiving LRS wasdetermined to have a 71% adhesion coverage using equation 2. Table 9shows the results obtained using the method discussed above for fucan 6.The results in the table below are shown as the reduction in adhesioncoverage relative to the control group.

Table 9 provides the result of treating eight uterine horns with fucan6.

TABLE 9 Increase in rabbit uterine horn adhesion using fucan 9 relativeto control LRS Number of % Reduction in uterine Dose Uterine hornadhesion (mg/kg) Horns coverage vs. control Fucan 5 8 −18% (i.e., 18%increase in fibrous 6 adhesions compared to control)

As can be seen from the results of Table 9, fucan 6 prepared using knownmethods and having a total non-fucan content of greater than 50% w/w ofthe fucan was not efficacious in the treatment of fibrous adhesions.

Example 22: Uterine Horn Fibrous Adhesion Treated with Fucan 7

To determine the efficacy of the purified/modified fucan 7 in inhibitingsurgical adhesions, the following double uterine horn (DUH) surgerieswere performed on both horns of a total of four New Zealand Whiterabbits. Prior to surgery, the rabbits were weighed and then preparedfor surgery by premedication with ketamine and xylazine.

Fucoidan solution was prepared at 0.33 mg/mL in Lactated RingersInjection USP (LRS), sterilizing by filtration. All instruments weresterile, and a sterile field was maintained throughout the surgeries.The abdomen was cleaned and entered via a midline abdominal incision.The uterine horns were located, exteriorized and scraped to inducedamage. The abdominal wall near the scraped uterine horns was alsoscraped. The damaged uterine horns and abdominal wall were placed nextto each other and stabilized with sutures. About 15 mL/kg fucoidansolution per rabbit weight was applied to the abdominal cavity beforethe incision was closed. Adhesion was evaluated two weeks after thesurgery. Three rabbits were evaluated at each fucoidan concentrationprepared. Length of the uterine horn adhesion was measured with a ruler.The uterine horn adhesion length was calculated using equation 2.

The same surgical method was applied to 4 New Zealand White rabbits,receiving about 15 mL/kg of control Lactated Ringer's Injection USP(LRS) instead of fucoidan solution. The control group receiving LRS wasdetermined to have a 76% adhesion coverage using equation 2. Table 10shows the results obtained using the method discussed above for fucan 7.The results in the table below are shown as the reduction in adhesioncoverage relative to the control group.

Table 10 provides the result of treating eight uterine horns with fucan7.

TABLE 10 Decrease in rabbit uterine horn adhesion using fucan 7 relativeto control LRS Number % Reduction in Dose of Uterine uterine hornadhesion (mg/kg) Horns coverage vs. control Fucan 5 8 3.7% (i.e., 3.7%reduction in fibrous 7 adhesions compared to control)

As can be seen from the results of Table 10, fucan 7 prepared usingknown methods and having a total non-fucan content of greater than 50%w/w of the fucan is not efficacious in the treatment of fibrousadhesions.

Example 23: Uterine Horn Fibrous Adhesion Treated with Fucan 4

To determine the efficacy of the purified/modified fucan 4 in inhibitingsurgical adhesions, the following double uterine horn (DUH) surgerieswere performed on both horns of a total of three New Zealand Whiterabbits. Prior to surgery, the rabbits were weighed and then preparedfor surgery by premedication with ketamine and xylazine.

Fucoidan solution was prepared at 5 mg/mL in Lactated Ringers InjectionUSP (LRS), sterilizing by filtration. All instruments were sterile, anda sterile field was maintained throughout the surgeries. The abdomen wascleaned and entered via a midline abdominal incision. The uterine hornswere located, exteriorized and scraped to induce damage. The abdominalwall near the scraped uterine horns was also scraped. The damageduterine horns and abdominal wall were placed next to each other andstabilized with sutures. The top third and the bottom third of themuscle incision was closed and 5 mL/kg fucoidan solution per rabbitweight was applied to the abdominal cavity. The muscle incision wastemporarily closed and the fucoidan solution left in the abdominalcavity for 30 minutes. The muscle incision was reopened and theabdominal cavity was flushed with 10 mL/kg LRS. The majority of thefluid in the abdominal cavity was suctioned out before the incision wasclosed. Adhesion formation was evaluated two weeks after the surgery.Length of the uterine horn adhesion was measured with a ruler. Theuterine horn adhesion coverage percentage, being the length of theadhesion as a percentage of the total damaged uterine horn length wascalculated using equation 2.

Table 11 shows the results obtained using the method discussed above forfucan 4, being a representative example of a purified/modified fucan.The results in the table below are shown as the mean adhesion lengthacross the 6 uterine horns scored.

Table 11 provides the results of treating six uterine horns with fucan4.

TABLE 11 Adhesion length using fucan 4 Dose Number of Mean % (mg/kg)Uterine Horns adhesion length Fucan 25 6 0% (i.e., no adhesions 4 werefound)

As can be seen from the results of Table 11, purified/modified fucanscan be used to successfully inhibit, prevent, remove, reduce, orotherwise treat post-surgical uterine horn adhesions.

Example 24: Uterine Horn Fibrous Adhesion Treated with aPurified/Modified Fucan Composition

To determine the efficacy of a purified/modified fucan compositioncomprising a total fucose, galactose, sulfate and counterions of 92% w/win inhibiting surgical adhesions, the following double uterine horn(DUH) surgeries were performed on both horns of a total of twenty NewZealand White rabbits. Prior to surgery, the rabbits were weighed andthen prepared for surgery by premedication with midazolam anddexmeditomidine.

Fucoidan solution was prepared at each concentration of 0.02 mg/mL, 0.1mg/mL, 0.5 mg/mL, or 2.5 mg/mL in Lactated Ringers Injection USP (LRS),sterilizing by filtration. All instruments were sterile, and a sterilefield was maintained throughout the surgeries. The abdomen was cleanedand entered via a midline abdominal incision. The uterine horns werelocated, exteriorized and scraped to induce damage. The abdominal wallnear the scraped uterine horns was also scraped. The damaged uterinehorns and abdominal wall were placed next to each other and stabilizedwith sutures. About 2 mL/kg fucoidan solution per rabbit weight wasapplied to the abdominal cavity before the incision was closed. Adhesionwas evaluated two weeks after the surgery. Five rabbits were treated andevaluated for each fucoidan concentration prepared. Length of theuterine horn adhesion was measured with a ruler. The uterine hornadhesion length was calculated using equation 2.

The same surgical method was applied to 5 additional New Zealand Whiterabbits for control, each receiving about 2 mL/kg of control LactatedRinger's Injection USP (LRS) instead of fucoidan solution. The controlgroup receiving LRS was determined to have a 100% adhesion coverageusing equation 2. Table 12 shows the results obtained using the methoddiscussed above for the purified/modified fucan composition at differentconcentrations and dosages (in total forty uterine horns were treated,10 each for each concentration of the purified/modified fucancomposition); the results are shown as the reduction in adhesioncoverage relative to the control group.

TABLE 12 Decrease in rabbit uterine horn adhesion using apurified/modified fucan composition relative to control LRS NumberConcen- of tration Dose Uterine % Reduction in uterine horn (mg/mL)(mg/kg) Horns adhesion coverage vs. control  0.02 0.04 10 10% (i.e., 10%decrease in fibrous adhesions compared to control) 0.1 0.2  10 30%(i.e., 30% decrease in fibrous adhesions compared to control) 0.5 1  1071% (i.e., 71% decrease in fibrous adhesions compared to control) 2.5 5 10 95% (i.e., 95% decrease in fibrous adhesions compared to control)

As can be seen from the results of Table 12, purified/modified fucancompositions can be used to successfully inhibit, prevent, remove,reduce, or otherwise treat post-surgical uterine horn adhesions.

REFERENCE NUMERALS LIST

-   1200 Cationic content modification system-   1202 Input supply line-   1204 Pre-filter-   1206 Cationic content modification system output valve-   1208 Cationic content modification system output line-   1210 Tangential flow filtration (TFF) filter-   1212 TFF supply line-   1214 TFF input pump-   1216 Fucan container-   1217 TFF retentate valve-   1218 TFF retentate return line-   1219 TFF permeate output line-   1220 First diafiltration solution container-   1224 First diafiltration solution valve-   1225 First diafiltration solution supply line-   1230 Second diafiltration solution container-   1234 Second diafiltration solution valve-   1235 Second diafiltration solution supply line

All terms used herein are used in accordance with their ordinarymeanings unless the context or definition clearly indicates otherwise.Also unless expressly indicated otherwise, in this disclosure the use of“or” includes “and” and vice-versa. Non-limiting terms are not to beconstrued as limiting unless expressly stated, or the context clearlyindicates, otherwise (for example, “including,” “having,” and“comprising” typically indicate “including without limitation”).Singular forms, including in the claims, such as “a,” “an,” and “the”include the plural reference unless expressly stated, or the contextclearly indicates otherwise.

Unless otherwise stated, adjectives herein such as “substantially” and“about” that modify a condition or relationship characteristic of afeature or features of an embodiment, indicate that the condition orcharacteristic is defined to within tolerances that are acceptable foroperation of the embodiment for an application for which it is intended.

The scope of the present methods, compositions, systems, etc., includesboth means plus function and step plus function concepts. However, theclaims are not to be interpreted as indicating a “means plus function”relationship unless the word “means” is specifically recited in a claim,and are to be interpreted as indicating a “means plus function”relationship where the word “means” is specifically recited in a claim.Similarly, the claims are not to be interpreted as indicating a “stepplus function” relationship unless the word “step” is specificallyrecited in a claim, and are to be interpreted as indicating a “step plusfunction” relationship where the word “step” is specifically recited ina claim.

From the foregoing, it will be appreciated that, although specificembodiments have been discussed herein for purposes of illustration,various modifications can be made without deviating from the spirit andscope of the discussion herein. Accordingly, the systems and methods,etc., include such modifications as well as all permutations andcombinations of the subject matter set forth herein and are not limitedexcept as by the appended claims or other claim having adequate supportin the discussion and figures herein.

What is claimed is:
 1. A purified and modified, medically acceptablefucan composition consisting of at least about 96% w/w purified andmodified fucans and no more than about 4% non-fucan components orcompounds or substances, wherein the purified and modified fucan musthave fucose, sulfate and counterions, and may further consist ofgalactose, glucose, rhamnose, mannose, xylose and glucuronic acid. 2.The purified and modified, medically acceptable fucan composition ofclaim 1 wherein the non-fucan components comprise water.
 3. The purifiedand modified, medically acceptable fucan composition of claim 1 whereinthe purified and modified, medically acceptable fucan compositionconsists of the purified and modified fucan and no more than about 3% ofthe non-fucan components.
 4. The purified and modified, medicallyacceptable fucan composition of claim 1 wherein the purified andmodified, medically acceptable fucan composition consists of thepurified and modified fucan and no more than about 2% of the non-fucancomponents.
 5. The purified and modified, medically acceptable fucancomposition of claim 1 wherein the purified and modified, medicallyacceptable fucan composition consists of the purified and modified fucanand no more than about 1% of the non-fucan components.
 6. The purifiedand modified, medically acceptable fucan composition of claim 1 whereinthe purified and modified, medically acceptable fucan compositionconsists of the purified and modified fucan and no more than about 0.1%of the non-fucan components.
 7. The purified and modified, medicallyacceptable fucan composition of any one of claim 1, 3, or 5 wherein thefucose content of the purified and modified fucan is greater than 25%w/w.
 8. The purified and modified, medically acceptable fucancomposition of any one of claim 1, 3 or 5 wherein the galactose contentof the purified and modified fucan is less than 10% w/w.
 9. The purifiedand modified, medically acceptable fucan composition of any one of claim1, 3, or 5 wherein the total counterion content of the purified andmodified fucan is less than 17% w/w.
 10. The purified and modified,medically acceptable fucan composition of any one of claim 1, 3 or 5wherein the counterion is a pharmaceutically acceptable counterion. 11.The purified and modified, medically acceptable fucan composition ofclaim 10 wherein the pharmaceutically acceptable counterion consistsessentially of at least one of sodium and potassium.
 12. The purifiedand modified, medically acceptable fucan composition of any one of claim1, 3 or 5 wherein the purified and modified fucan has a molecular weightdistribution wherein at least 60% w/w of the distribution is greaterthan 100 kDa when measured using an aqueous gel permeationchromatography set up consisting essentially of: one 300 mm analyticalgel permeation chromatography column with a 7.8 mm inner diameter packedwith hydroxylated polymethacrylate-based gel, having an effectivemolecular weight range of between about 50 kDa and about 5,000 kDa, one300 mm analytical gel permeation chromatography column with a 7.8 mminner diameter packed with hydroxylated polymethacrylate-based gel,having an effective molecular weight range of between about 1 kDa andabout 6,000 kDa and one 40 mm guard column with a 6 mm inner diameterpacked with hydroxylated polymethacrylate-based gel, the two analyticalgel permeation chromatography columns and the one guard column containedin a column compartment at about 30° C.; a refractive index detector atabout 30° C.; 0.1M sodium nitrate mobile phase run at 0.6 mL/min; andquantification against a peak molecular weight standard curve consistingessentially of a first dextran standard with a peak molecular weight ofabout 2,200 kDa, a second dextran standard with a peak molecular weightof between about 720 kDa and about 760 kDa, a third dextran standardwith a peak molecular weight between about 470 kDa and about 510 kDa, afourth dextran standard with a peak molecular weight between about 370kDa and about 410 kDa, a fifth dextran standard with a peak molecularweight between about 180 kDa and about 220 kDa, and a sixth dextranstandard with a peak molecular weight between about 40 kDa and 55 kDa.13. The purified and modified, medically acceptable fucan composition ofclaim 12, wherein the purified and modified fucan has a molecular weightdistribution wherein at least 92% w/w of the distribution is greaterthan 100 kDa.
 14. The purified and modified, medically acceptable fucancomposition of claim 12, wherein the purified and modified fucan has aweight average molecular weight greater than 100 kDa.
 15. The purifiedand modified, medically acceptable fucan composition of any one of claim1, 3, or 5, wherein the purified and modified fucan has a sulfationlevel of between about 20% w/w and 60% w/w.
 16. The purified andmodified, medically acceptable fucan composition of any one of claim 1,3 or 5, wherein the purified and modified fucan has a total carbohydratecontent between 27% w/w and 80% w/w.
 17. The purified and modified,medically acceptable fucan composition of claim 16, wherein the purifiedand modified fucan has a total of glucuronic acid, mannose, rhamnose,glucose and xylose content as a percentage of the total carbohydratecontent below about 12% w/w.
 18. The purified and modified, medicallyacceptable fucan composition of any one of claim 1, 3 or 5 wherein thepurified and modified, medically acceptable fucan composition whendissolved in water at a concentration of 50 mg/mL has a viscosity ofbetween about 4 cP and 50 cP.
 19. The purified and modified, medicallyacceptable fucan composition of any one of claim 1, 3 or 5 wherein thepurified and modified, medically acceptable fucan composition is a whitesolid.
 20. The purified and modified, medically acceptable fucancomposition of any one of claim 1, 3 or 5 wherein the purified andmodified, medically acceptable fucan composition when dissolved in waterat a concentration from 1 mg/mL through 100 mg/mL forms a solution thatis clear and colorless.
 21. The purified and modified, medicallyacceptable fucan composition of any one of claim 1, 3 or 5 wherein thepurified and modified, medically acceptable fucan comprises less than 5%w/w acetyl content.
 22. The purified and modified, medically acceptablefucan composition of any one of claim 1, 3 or 5 wherein the purified andmodified fucan comprises an acetyl content of substantially 0% w/w whenmeasured by 2D 1H-13C heteronuclear multiple quantum coherence at 70° C.with solvent signal suppression on a 600 MHz spectrometer equipped with5-mm cold probe, in the range from 10-30 ppm in the carbon dimension, in8 increments of 256-512 scans each.
 23. A purified and modified,medically acceptable composition comprising a therapeutically effectiveamount of the purified and modified, medically acceptable fucancomposition of any one of claim 1, 3 or
 5. 24. A medical compositioncomprising between about 0.02 mg/mL and 100 mg/mL of the purified andmodified, medically acceptable fucan composition of any one of claim 1,3 or 5 in a medically acceptable liquid, wherein the medical compositionis configured and composed to treat a disease or condition in an animal.25. The medical composition of claim 24 wherein the disease or conditionis a fibrous adhesion.
 26. The purified and modified, medicallyacceptable fucan composition of claim 10 wherein the pharmaceuticallyacceptable counterion consists of at least one of calcium and magnesium.27. The purified and modified, medically acceptable fucan composition ofclaim 12 wherein the purified and modified fucan has a sulfation levelof between about 20% w/w and 60% w/w.
 28. The purified and modified,medically acceptable fucan composition of claim 12, wherein the purifiedand modified fucan has a total carbohydrate content between 27% w/w and80% w/w.
 29. The purified and modified, medically acceptable fucancomposition of claim 15, wherein the purified and modified fucan has atotal carbohydrate content between 27% w/w and 80% w/w.
 30. The purifiedand modified, medically acceptable fucan composition of claim 27,wherein the purified and modified fucan has a total carbohydrate contentbetween 27% w/w and 80% w/w.
 31. The purified and modified, medicallyacceptable fucan composition of claim 12 wherein the purified andmodified fucan comprises less than 5% w/w acetyl content.
 32. Thepurified and modified, medically acceptable fucan composition of claim15 wherein the purified and modified fucan comprises less than 5% w/wacetyl content.
 33. The purified and modified, medically acceptablefucan composition of claim 27 wherein the purified and modified fucancomprises less than 5% w/w acetyl content.
 34. The purified andmodified, medically acceptable fucan composition of claim 30 wherein thepurified and modified fucan comprises less than 5% w/w acetyl content.